TYPE Review

PUBLISHED 11 November 2024

DOI 10.3389/fcell.2024.1435461

Navigating stem cell culture:

OPEN ACCESS insights, techniques, challenges,
EDITED BY
Ming Pei,
West Virginia University, United States
and prospects
REVIEWED BY
Sandeep P. Dumbali, Aleksandra Górska 1, Mateusz Trubalski 2, Bartosz Borowski 2,
Moffitt Cancer Center, United States
Anastasia Conti,
Adam Brachet 3, Sylwia Szymańczyk 4* and Renata Markiewicz 5*
San Raffaele Telethon Institute for Gene 1
Department of Normal, Clinical and Imaging Anatomy, Medical University of Lublin, Lublin, Poland,
Therapy (SR-Tiget), Italy 2
Students Scientific Association, Department of Normal, Clinical and Imaging Anatomy, Medical
*CORRESPONDENCE University of Lublin, Lublin, Poland, 3Student Scientific Association, Department of Forensic Medicine,
Renata Markiewicz, Medical University of Lublin, Lublin, Poland, 4Department of Animal Physiology, Faculty of Veterinary
[email protected] Medicine, University of Life Sciences in Lublin, Lublin, Poland, 5Occupational Therapy Laboratory, Chair
Sylwia Szymańczyk, of Nursing Development, Medical University of Lublin, Lublin, Poland
[email protected]

RECEIVED 03 June 2024

ACCEPTED 09 October 2024
PUBLISHED 11 November 2024 Stem cell research holds huge promise for regenerative medicine and disease
CITATION modeling, making the understanding and optimization of stem cell culture a
Górska A, Trubalski M, Borowski B, Brachet A, critical aspect of advancing these therapeutic applications. This comprehensive
Szymańczyk S and Markiewicz R (2024)
Navigating stem cell culture: insights, review provides an in-depth overview of stem cell culture, including general
techniques, challenges, and prospects. information, contemporary techniques, encountered problems, and future
Front. Cell Dev. Biol. 12:1435461. perspectives. The article begins by explaining the fundamental characteristics
doi: 10.3389/fcell.2024.1435461
of various stem cell types, elucidating the importance of proper culture
COPYRIGHT
conditions in maintaining pluripotency or lineage commitment. A detailed
© 2024 Górska, Trubalski, Borowski, Brachet,
Szymańczyk and Markiewicz. This is an open- exploration of established culture techniques sheds light on the evolving
access article distributed under the terms of the landscape of stem cell culture methodologies. Common challenges such as
Creative Commons Attribution License (CC BY). genetic stability, heterogeneity, and differentiation efficiency are thoroughly
The use, distribution or reproduction in other
forums is permitted, provided the original discussed, with insights into cutting-edge strategies and technologies aimed
author(s) and the copyright owner(s) are at addressing these hurdles. Moreover, the article delves into the impact of
credited and that the original publication in this substrate materials, culture media components, and biophysical cues on stem
journal is cited, in accordance with accepted
academic practice. No use, distribution or cell behavior, emphasizing the intricate interplay between the microenvironment
reproduction is permitted which does not and cell fate decisions. As stem cell research advances, ethical considerations and
comply with these terms. regulatory frameworks become increasingly important, prompting a critical
examination of these aspects in the context of culture practices. Lastly, the
article explores emerging perspectives, including the integration of artificial
intelligence and machine learning in optimizing culture conditions, and the
potential applications of stem cell-derived products in personalized medicine.
This comprehensive overview aims to serve as a valuable resource for researchers
and clinicians, fostering a deeper understanding of stem cell culture and its key
role in advancing regenerative medicine and biomedical research.

KEYWORDS

stem, cells, culture, methods, pluripotency, perspectives

Introduction
In the dynamic field of stem cell research, the evolution of culture methodologies plays a
pivotal role in unlocking the vast therapeutic and scientific potential inherent in these
remarkable cells. This article provides a comprehensive overview, synthesizing key findings
from seminal studies to offer a nuanced exploration of general information, techniques,
persistent challenges, and future perspectives in the domain of stem cell culture. The past

Frontiers in Cell and Developmental Biology 01 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

TABLE 1 Comparison of 2D and 3D cultures (Raitanen et al., 2023; Souza et al., 2018; Ylostalo, 2020; Baharvand et al., 2006).

Criterion 2D cell culture system 3D cell culture system

Cell Structure Monolayer of cells on a flat surface Three-dimensional structures with cell aggregates

Culturing Environment Flat surface (e.g., Petri dish) Gel matrices, microspheres, or bioreactors

Cell Interactions Limited, primarily surface interactions Fully three-dimensional, more complex interactions

In Vivo Representation Limited, less realistic Better simulation of in vitro environment

Phenotypic Impact May lead to altered cell properties Better retention of in vitro cell properties

Metabolic Efficiency Often less representative Typically better, more akin to natural conditions

Applications Rapid and cost-effective assays, basic research Differentiation studies, therapies, tissue engineering

Technical Requirements Lower, easier to use Higher, requires more advanced technologies

decade has witnessed groundbreaking discoveries that have offering a comprehensive and up-to-date exploration of stem cell
reshaped our understanding of stem cells (Chen et al., 2014). The culture to optimize methodologies, propelling the field towards its
work of Takahashi and Yamanaka (Takahashi and Yamanaka, 2013) full potential in biomedical research.
introduced induced pluripotent stem cells (iPSCs), a transformative
concept that facilitated the reprogramming of somatic cells into a
pluripotent state, bypassing ethical concerns associated with General information about stem cells
embryonic stem cells (Aboul-Soud et al., 2021; Yamanaka, 2020;
Karagiannis et al., 2019; Liu et al., 2020). Concurrently, the In the context of developmental ontogenesis, stem cells (SCs)
investigations furthered our understanding of human embryonic can be broadly classified into two principal categories: embryonic
stem cells (hESCs), contributing to the establishment of robust stem cells (ESCs), isolated from the inner cell mass of blastocysts,
culture protocols for these cells (Kobold et al., 2023). and adult stem cells (ASCs), colloquially referred to as somatic stem
Advancements in stem cell culture techniques have been cells (SSCs), distributed across various adult tissues (Slack, 2018;
instrumental in harnessing the full potential of these cells for Gao et al., 2018). Embryonic stem cells (ESCs), characterized by
therapeutic applications. Research by Chen et al. (2019) their self-renewal capacity, embody pluripotency, originating from
emphasized the importance of three-dimensional (3D) culture the inner cell mass (ICM) of developing blastocysts. Pluripotency
systems, demonstrating their superiority in maintaining stem cell signifies the singular cell’s capability to engender all cellular lineages
pluripotency and enhancing differentiation potential compared to throughout both embryonic and adult organismal development.
traditional two-dimensional (2D) approaches (Ylostalo, 2020). A Tissue-specific stem cells orchestrate the dynamic turnover of
comparison of 2D and 3D cultures can be found in Table 1. distinct tissue phenotypes in mammals and other metazoans
Additionally, the recent article of Yu et al. (2023) showcased the (Young, 2011; Christopher and Baker, 2018). Their ubiquitous
integration of microfluidic platforms to precisely control the stem presence spans a diverse array of tissues, including but not
cell microenvironment, influencing cellular responses in limited to bone marrow (BMSCs), adipose tissue (ADSCs), dental
unprecedented ways. However, the translation of stem cell pulp (DPSCs), blood (HSCs), amniotic fluid (AFSCs), umbilical cord
research into clinical applications is not without its challenges (UCSCs), and a sundry of other tissues. Despite sharing a vernacular
(McKee and Chaudhry, 2017). There is the persistent issue of nomenclature, these classifications reveal myriad distinctions (Zeyu
genetic instability in long-term cultures, necessitating a nuanced et al., 2023; Orticelli et al., 2021).
approach to mitigate risks associated with genomic alterations Segregated according to their differentiative potential, stem cells
(Prieto Gonzalez, 2022). Furthermore, the intrinsic heterogeneity delineate into totipotent SCs, pluripotent SCs (PSCs), multipotent
within stem cell populations is a major hurdle, demanding SCs, and unipotent SCs (Gao et al., 2018). Totipotent cells,
innovative strategies to enhance the homogeneity of cell represented by the zygote and early blastomeres, possess the
populations for therapeutic efficacy (Mas-Bargues and Borrás, exceptional ability to give rise to all cellular lineages, playing a
2021; Wang et al., 2021a). Looking ahead, the article explores central role in initiating organismal development (Maemura et al.,
emerging perspectives and potential solutions to current 2021). Conversely, pluripotent cells, found in the inner cell mass of
challenges. Recent investigations showcase the integration of blastocysts, exhibit a broad capacity to generate all cell types, except
artificial intelligence (AI) and machine learning in optimizing those specific to the extraembryonic trophoblast lineage. This
stem cell culture conditions, promising data-driven insights for distinction underscores the critical roles these cell types play in
enhanced reproducibility and efficiency (Nosrati and Nosrati, the early stages of life, with totipotent cells guiding the foundational
2023). Additionally, Li et al. (2018) discusses the transformative steps of complete organism formation, and pluripotent cells
potential of stem cell-derived products in personalized medicine, contributing to the differentiation of diverse cell types crucial for
providing a glimpse into a future where tailored therapeutic embryonic development (Cai et al., 2022; Malik and Wang, 2022;
interventions are based on individual patient profiles (Li et al., Sobhani et al., 2017). Multipotent stem cells manifest the ability to
2018). This review aims to consolidate the wealth of knowledge differentiate into all cell types from a single germ layer, while

Frontiers in Cell and Developmental Biology 02 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

FIGURE 1
Sources of stem cells.

unipotent stem cells are defined by their competence to differentiate et al., 2012). Tissues characterized by a high rate of cellular turnover,
solely into one lineage (Kolios and Moodley, 2013). Oligopotent such as blood and gut epithelium, harbor populations of incessantly
stem cells, distinguished by their unique capacity for self-renewal, proliferating adult stem cells and progenitor cells, adept at
demonstrate the remarkable ability to initiate and sustain two or generating differentiating progeny. Additionally, many tissues
more distinct lineages within a specific tissue. An exemplary maintain populations of stem cells in a quiescent state, either to
illustration of oligopotent stem cells can be observed in provide support for cycling progenitors or to serve as a reserve for
hematopoietic stem cells, known for their versatility in potential tissue injuries (de Morree and Rando, 2023). A pivotal
differentiating into both myeloid and lymphoid lineages, thereby prerequisite for a stem cell is the demonstration of clonality and the
contributing to the diverse cellular composition of the blood (Cheng capacity to generate a myriad of functional cells. The ‘self-renewing’
et al., 2020). attributes of a stem cell may manifest through symmetrical division,
In the context of pulmonary biology, ongoing investigations yielding exclusively either stem cells or all progenitor cells, or
propose a fascinating possibility: bronchoalveolar duct junction through asymmetrical division, capable of producing both stem
cells, identified as oligopotent, may serve as the originators of cells and progenitor cells (Sanders et al., 2006).
both bronchiolar epithelium and alveolar epithelium. This
intriguing finding underscores the dynamic nature of oligopotent
stem cells in orchestrating tissue-specific regeneration and Sources of stem cells
underscores their role in maintaining the intricate cellular
architecture of the respiratory system (Ilic and Polak, 2011). In the realm of regenerative medicine and cellular therapeutics,
The phenomenon of continuous regeneration serves as an stem cells are procured from various biological reservoirs,
intrinsic process orchestrated by stem cells, playing a pivotal role encompassing four fundamental sources. These encompass (1)
in maintaining homeostasis within multicellular organisms. embryonic tissues, (2) fetal tissues such as the fetus, placental
Senescent, functionally specialized mature cells within adult components (amnion and chorion), amniotic fluid, and umbilical
organs are perpetually supplanted by newly generated cells (Lee cord structures (Wharton’s jelly and blood), (3) specific niches
and Hong, 2020). This cellular turnover process is notably within the adult organism, notably adipose tissue, bone marrow,
heightened within the hematopoietic system, intestinal skeletal muscle, and blood, and (4) differentiated somatic cells
epithelium, or epidermis, in stark contrast to the more measured subjected to genetic reprogramming, exemplified by induced
pace observed in skeletal muscles, liver, or cardiac tissues (Ratajczak pluripotent stem cells (iPSCs) (Figure 1.) (Bacakova et al., 2018;

Frontiers in Cell and Developmental Biology 03 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

Mushahary et al., 2018; Lovell-Badge et al., 2021). Historically, bone enzymatic methodology (Priya et al., 2014). The amalgamation of
marrow (BM) has stood as the preeminent reservoir of these methodologies underscores a nuanced approach to
mesenchymal stem cells (MSCs) in the human context. Despite establishing reliable, robust, and standardized MSC isolation
its richness in hematopoietic stem cells, BM harbors only a scant protocols, exemplifying the potential of UC and AT as highly
population of MSCs. Moreover, the arduous and anesthesia- relevant and promising tissue sources for MSCs (Mushahary
requiring BM harvesting procedure has restricted the utilization et al., 2018).
of BM-derived MSCs (bmMSCs) in both investigative and clinical Recent improvements in culturing hematopoietic stem cells
settings. Presently, MSCs are isolatable from diverse tissue sources, (HSCs) focus on enhancing their ex vivo expansion and
including dental tissues, integumentary structures, salivary glands, maintaining their functionality over extended periods. Advances
limb buds, and menstrual blood (Rodríguez-Fuentes et al., 2021). include the development of fully defined, albumin-free culture
Adipose tissue (AT) emerges as a salient source of MSCs, systems that eliminate the variability of traditional supplements
characterized by its ubiquity and accessibility, rendering adipose- like bovine serum albumin. These new methods allow for substantial
derived MSCs (adMSCs) pivotal candidates for autologous and expansion of HSCs, with fold increases reaching several hundred
allogeneic stem cell-based therapies and tissue engineering times over a month. Improved culture conditions also support clonal
(Palumbo et al., 2018). In contradistinction, perinatal tissues, analysis of HSC heterogeneity and enable transplantation into
such as the amnion, chorion, and umbilical cord (UC), represent nonconditioned recipients, offering new potential for research
promising repositories of MSCs, given the advantageous attributes and therapeutic applications in hematopoiesis and immune
conferred by the youthfulness of the donors. While MSC derivation system studies (Wilkinson et al., 2020).
from various perinatal tissues is feasible, UC tissue, specifically Current improvements in culturing mesenchymal stromal cells
Wharton’s jelly, emerges as a superior MSC reservoir when (MSCs) have focused on optimizing growth conditions to enhance
compared to umbilical cord blood (UCB) (Ferrúa et al., 2017; their expansion, viability, and therapeutic potential. Key
Chu et al., 2020). advancements include the use of serum-free or xeno-free media,
The acquisition of stem cells is predicated upon two principal which eliminates animal-derived components and reduces
methodologies: enzymatic isolation and explant culturing. variability in culture outcomes. Newer bioreactor systems and 3D
Enzymatic isolation procedures are contingent upon the source culture methods have also been developed, enabling more efficient
of MSCs, necessitating tailored techniques, specific media large-scale expansion while preserving MSC functionality.
formulations, serum constituents, and cell surface markers Additionally, refined protocols for maintaining MSC stemness,
(Bacakova et al., 2018; Mushahary et al., 2018). In the explant differentiation potential, and immunomodulatory properties have
method, devoid of enzymatic intervention, the original tissue is contributed to their increased use in regenerative medicine and cell-
sectioned into smaller fragments, subsequently arranged in culture based therapies (Todtenhaupt et al., 2023).
dishes or flasks. After this, cells commence migration from the tissue
fragments, adhering to the culture surface (Hendijani, 2017). The
explant methodology is notable for its proclivity to generate cell The employment of stem cell culture in
populations characterized by reduced heterogeneity, resulting in therapy and science (cell lines)
augmented proliferation rates and enhanced cell viability, as
compared to the enzymatic approach (Chu et al., 2020). This The growing interest in culturing stem cells excited many
inclination is likely attributable to the presence of integral tissue researchers all around the world to better understand the
fragments and undissociated extracellular matrix (ECM) during the processes occurring in the cultures development and to predict
explant culture process. These components contribute to the the overall possibilities of these cells’ usage. Throughout recent
establishment of a protective milieu, mitigating the impact of years, many milestones have been reached in this field, and the use of
proteolytic and mechanical stressors and thereby creating an stem cells cultures continues to grow and pins researchers’ faith on
environment conducive to the migration of cells (Sajeesh et al., applying it in more treatment tactics and scientific research. Many
2020). While enzymatic digestion facilitates the isolation of research works are conducted using rodent stem cells (especially
fibroblast-like (stem) cells and the concomitant liberation of mouse ones), and the outcomes of these works suggest that cultured
endothelial cells and pericytes, the explant culture paradigm stem cells can help in the treatment of joints diseases (e.g.,
offers additional advantages. These include the release of rheumatoid arthritis), peritonitis, colitis, and many more (Yen
cytokines and growth factors into the culture medium, an et al., 2023; Lan et al., 2021; Marega et al., 2023; Zaripova
augmented yield of stromal cells, a truncated proliferation et al., 2023).
timeframe, and the simultaneous expression of surface markers Therapeutic properties of stem cell cultures can be helpful for
CD73, CD90, and CD105, coupled with the conspicuous absence various specialists. Dierick et al. (2021) points out that multipotent
of CD14, CD31, CD34, and CD45 in all MSC populations (Lu et al., stem cells (MSC) are currently under consideration as a possible
2021). Quantitative assessments predicated on nucleated cell treatment of pulmonary hypertension (Dierick et al., 2021). Jiang
numbers per unit tissue weight underscore that the explant and Tuan (2023) in his work describes various types of stem cells
culture methodology confers a superior yield of stromal cells in used in creating a cartilage-like tissue in human joints, which
comparison to the enzymatic method. This observed advantage may improves the quality of life of patients affected with arthropathies
stem from diminished cellular adhesion following exposure to (Jiang and Tuan, 2023; Giorgino et al., 2023; Hwang et al., 2021).
enzymatic treatment and the concomitant loss of cells during the One of the yet-to-overcome challenge remains employing stem cells
sequential procedures of filtration and washing inherent in the to repair infarcted cardiac tissue. Cardiovascular diseases remain

Frontiers in Cell and Developmental Biology 04 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

FIGURE 2
Stem cells culturing process.

one of the most common deaths causes all around the world, and the researchers, Takahashi and Yamanaka (2006), discovered how to
possibility of heart regeneration is very promising (Parrotta et al., turn a multipotent stem cell into a pluripotent one (Takahashi and
2019; Campostrini et al., 2021; Barreto et al., 2019). The researchers Yamanaka, 2006). This has been one of the milestones in the stem
are also examining the usage of cell lines, which are induced into cells culturing, as it enabled using these cells in human therapy, as
male germ-like cells, in the treatment of male infertility the cells obtained in this process are biocompatible with the receiver.
(Malekmohamadi et al., 2019). Chen et al. (2020) in his work There are several ways to culture stem cells, depending on the
presents the idea of forming a healthy liver, using 3D stem cells, source of the cells themselves. In this work, there will be highlighted
which would be transplanted to patients suffering from end-stage two methods of culturing stem cells (Figure 2.). Human embryonic
liver diseases, such as liver cancer (Chen et al., 2020). Scientists also stem cells (hESCs), the first type of stem cells, can be obtained from
utilize stem cells in the process of understanding mechanisms that the preimplantation embryos, more precisely-the blastocyst
occur in tumors, such as glioblastoma (Nikitin et al., (Zakrzewski et al., 2019). First, the hESCs need to be separated
2023) and more. from the blastocyst (which can be done in the early stages of embryo
It is worth underlining that the increasing knowledge regarding differentiation, as it is safer for the embryo, or in vitro). There are
stem cell culture and its application in the modern therapeutic several methods to perform this procedure. The simplest one is the
approaches is possible due to discovering new technologies, better manual separation. The other possibilities consist of using enzymes’
understanding the processes taking place in stem cells, and the inhibitors, such as checkpoint kinase-1 (CHK1) inhibitor (Ware
growing interest in molecular science. Apart from medicine, stem et al., 2023), trypsin or ethylenediaminetetraacetic acid (EDTA)
cells also find use in other sciences, such as ecology. Xie et al. (2020) (Zakrzewski et al., 2019). After the separation is done, the obtained
in his work points out that obtaining these cells from various cells need to be placed in a special environment, containing nutrients
endangered fish species can help preserving them in the and growth factors, delivered daily. This mixture consists of such
environment (Xie et al., 2020). substances as antibiotics, insulin, transferrin, heparin, source of
sodium, fibroblast growth factor-4 (FGF-4), and many more
(Zhao et al., 2023). The culture will continue to grow, and with
The methods of culturing stem cells adequate factors, can be directed into more specified cells
(Zakrzewski et al., 2019).
The process of developing a stem cells culture has been Moreover, the microenvironment plays a critical role in stem cell
improving and changing for several years. In 2006, the Japanese behavior, particularly in regulating stem cell fate. Factors such as

Frontiers in Cell and Developmental Biology 05 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

extracellular matrix (ECM) composition, stiffness, and mechanical caused by common problems in cell culture, such as cross-
forces are key in determining the differentiation pathways of stem contamination between different species or within the same
cells. For instance, variations in ECM stiffness can direct stem cells species, misidentification of cells, genetic drift, bacterial, fungal,
toward specific lineages; softer matrices tend to promote neurogenic yeast, viral, or chemical contamination, and the lack of strict quality
differentiation, while stiffer matrices support osteogenic control testing (Nelson-Rees and Flandermeyer, 1976). Rough
differentiation. Mechanical forces like wall shear stress and estimates suggest that 16.1% of studies that were published used
circumferential strain also significantly influence stem cell fate by cell lines that had problems (Babic et al., 2019). It has been found
modulating cellular signaling pathways and gene expression. These that adding antibiotics and anti-mycotic to cell culture media helps
mechanical cues, in combination with the biochemical environment, reduce biological contamination from mycoplasma, yeast,
create a complex microenvironment that ultimately governs stem bacterium, and fungus. These drugs usually work by preventing
cell behavior and differentiation (Discher et al., 2005; Engler the formation of bacterial initiation complexes between mRNA and
et al., 2006). the bacterial ribosome (e.g., streptomycin), inhibiting the synthesis
After the differentiation, hESCs become multipotent, meaning of cell walls (e.g., penicillin), or compromising membrane
that they can only develop into specified germ layer (ectoderm, permeability (e.g., amphotericin B). However, prolonged usage of
endoderm, mesoderm). However, the earlier-mentioned discovery antibiotics may result in the sluggish growth rates of resistant or
of Yamanaka and Takahashi allows to undo this process and acquire persistent bacterial pollutants, which may cause subtle alterations in
a pluripotent cell from a multipotent one. These stem cells are called cell behavior and differentiation (Llobet et al., 2015; American Type
induced pluripotent stem cells (iPSCs). Culture Collection Standards Development Organization
To obtain iPSCs, mature cells, such as fibroblasts, epithelial cells, Workgroup ASN-0002, 2010). Therefore, to minimize bacterial
keratinocytes et cetera are drawn from the organism (Zakrzewski contamination in cell culture, researchers should endeavor to
et al., 2019). Later, these cells need to be reprogrammed by forcing maintain rigorous aseptic working conditions and refrain from
an expression of oncogenes. To test whether obtained iPSCs have using antibiotics in cell culture on a permanent basis (Ryu et al.,
sufficient reproductive properties, a specific test needs to be 2017; Varghese et al., 2017). In cell cultures, contamination can
conducted-the teratoma formation assay (Zakrzewski et al., come from chemical and biological sources. Indicators of this
2019). In this test, the iPSCs are injected into a rodent, and start contamination typically include sluggish cell growth,
to form a teratoma. Further on, the tumor is tested to determine morphological abnormalities, abrupt pH changes in the media,
which types of tissues has it formed. The outcome shows how active and higher concentrations of dead or floating cells in the culture.
they obtained iPSCs are. Some scientists also use the teratoma Regular screening for these pollutants in cell cultures is crucial to
formation to work on the sourced tissues, after collecting the guarantee consistency in results and prevent potentially harmful
tumor from the rodent (Lee et al., 2020). consequences.
It is necessary to mention the efforts from Dr. Raimondi’s lab to Different cell types may require specific culture vessel coatings
develop faithful 3D models to preserve stem cell in a more or substrates for optimal attachment and growth. Using
physiological context (Donnaloja et al., 2020; Steimberg et al., inappropriate surfaces can result in poor cell adhesion and
2020; Rey et al., 2020; Nava et al., 2012; Boeri et al., 2019). These viability. Primary cells can be more sensitive than established cell
models aim to mimic the natural stem cell niche more accurately lines. Attention must also be paid to specific requirements, such as
than traditional 2D cultures, promoting more realistic cell behavior, shorter passage intervals, specialized media, and unique culture
differentiation, and self-renewal. Advances include the use of conditions.
biomimetic scaffolds, hydrogels, and organoids that replicate the In biomedical research, cell culture is essential, but dependability
physical, chemical, and mechanical properties of the in vivo is compromised by problems including contamination and
environment. These 3D systems improve the maintenance of misidentification. Authentication is aided by resources such as
stem cell stemness and functionality, providing more reliable International Cell Line Authentication Committee (ICLAC) and
platforms for studying cell biology, disease modeling, and required testing. Adopting best practices, training employees, and
potential therapeutic applications. utilizing modern innovations like patient-derived organoids (PDOs)
There is much yet to be discovered in culturing stem cells. Each and patient-derived xenograft (PDXs) are all necessary to ensure
year novel methods are discovered, and the therapeutic and research safety. Reliance on animals is decreased by the rapid production of
properties of stem cells are amplified. patient-derived cell cultures made possible by CRISPR technology.
Continuous training of personnel is essential for a dependable cell
culture facility.
Problems and most common mistakes
in cell culture management
Decellularized extracellular matrix
Cell culture is a crucial technique in biological research, and
proper management is essential to ensure reliable and reproducible Decellularized extracellular matrix (dECM) derived from
results. Errors can occur in cell culture studies if they are not done cells has emerged as a powerful tool in stem cell culture,
correctly. To ensure the repeatability of in vitro research, it is crucial offering a variety of benefits that enhance the growth,
that cell culture investigations be carried out using good cell culture differentiation, and overall functionality of stem cells. The
practice (GCCP) (Pamies et al., 2022). The abundance of erroneous dECM is a scaffold composed of proteins, glycoproteins, and
and irreproducible results in the scientific literature is specifically other components that mimic the natural extracellular

Frontiers in Cell and Developmental Biology 06 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

Type Characterization Ref.

Viruses Due to the lack of impact on cellular growth, detecting viral contamination Schroder et al. (2022), Gombold et al. (2014), Meten (2002),
can be challenging. The minute size of these viruses (approximately 100 nm Geraghty et al. (2014), Borojevic et al. (1985)
in diameter) renders them invisible under a bright-field microscope.
Although cytopathic virus infections may not influence cellular growth,
they can still compromise the integrity of the culture. Furthermore,
laboratory personnel working with virally infected cell cultures may be at
risk of health hazards

Mycoplasmas Mycoplasmas are spherical to filamentous cells that lack intracytoplasmic Drexler and Uphoff (2002), Rottem, 2003; Into et al. (2004)
membranes and cell walls. With a diameter of about 300 nm, they are the
smallest self-replicating organisms. Mycoplasma infections can alter various
cellular activities of the host culture, including growth, metabolism,
migration, morphology, and responsiveness to growth factors.
Additionally, certain mycoplasma species may cause chromosomal damage
and abnormalities in the data

Bacteria The sizes and shapes of bacteria vary greatly, ranging from 0.5 to 20 µm. In Taur et al. (2014), Bartfeld et al. (2015)
cell culture conditions, bacterial pollutants can spread and colonize rapidly.
When tiny, moving granules appear between cells a few days after initial
contamination, microscopy is typically able to detect such contamination

Yeast and mold Fungi, including yeast cells, multiply more quickly than cells found in Puerta-Alcalde and Garcia-Vidal (2021), Otto and Green
mammals. They are typically 3–4 µm in size, although they can grow up to (2020)
40 µm. Within two to 3 days, contamination can be easily detected by
microscopic analysis or by changing the medium’s color. Notably, yeast is
not harmed by antibiotics such as streptomycin and penicillin

Parasites Certain precautions may need to be taken when handling freshly generated Herwaldt (2001), Herbert et al. (1980)
primary cell cultures from a donor organism that is suspected or confirmed
to be infected with intracellular protozoan parasites (such as Toxoplasma
gondii, Trypanosoma cruzi, Leishmania spp., Cryptosporidium parvum, or
Plasmodium spp.). It is important to take additional safety measures, such
as wearing protective gear and clothes. Needles and other sharp things
should not be used when working with parasite-infected cell lines to reduce
potential dangers

Prions Prions are difficult to inactivate since they are primarily made up of the Avar et al. (2020), Chou et al. (2015)
protein PrPSc and lack nucleic acids. Although most cell lines are resistant
to prion infection, some susceptible cell lines allow prions to replicate
steadily and permanently. These prions could be added to cell culture
medium that has been supplemented with bovine serum. Interestingly,
prions are notoriously hard to inactivate

Chemical, biological, and other Optimal cell growth can be negatively impacted by endotoxins/ Nims and Price (2017), Grzelak et al. (2001), Zigler et al.
nonliving contaminants lipopolysaccharides, detergents, metals, hormones, growth factors, (1985)
disinfection and cleaning agent residues, plasticizers, and other
contaminants. Water, sera, contaminated reagents, and specific culture
supplements can all lead to chemical contamination. Cell cultures might
also be jeopardized by impurities such as detergent residues or residues on
storage containers, glasses, pipettes, or tools used during disinfection.
Plasticizers may leak out of storage bottles and plastic tubing. Prolonged
exposure to visible or fluorescent light can produce free radicals by
photoactivating substances like riboflavin, tryptophan, or buffering agents
like HEPES and PIPES.

Inter- and intra-species cross- Cross-contamination in cell lines is both frequent and substantial. Inter- Capes-Davis et al. (2010), Huang et al. (2017), Weiskirchen
contamination and intra-species cross-contamination can originate from various sources, (2022)
including aerosol transmission, the use of unfiltered pipettes, shared media
and reagents between different cell lines, and the application of conditioned
materials

environment, providing crucial signals and structural support to proliferation, differentiation, and migration. These cues are
cultured cells. Here are the key benefits of using cell-derived difficult to replicate using synthetic materials.
dECM in stem cell culture (Wang et al., 2021b; Pei, 2017; Kim • Structural Support: The natural architecture of dECM offers
et al., 2018; Yao et al., 2019; Zhang et al., 2021): a physical framework that closely resembles the in vivo
environment, supporting proper cell attachment,
1. Mimicking the Native Microenvironment organization, and growth.
• Biochemical Cues: The dECM retains bioactive molecules 2. Enhanced Stem Cell Differentiation
from the original tissue, which can provide essential • Tissue-Specific Differentiation: dECM derived from specific
biochemical cues that guide stem cell behavior, including tissues can induce lineage-specific differentiation of stem

Frontiers in Cell and Developmental Biology 07 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

cells. For instance, cardiac dECM can promote cardiac These advantages make dECM a valuable tool in advancing stem cell
lineage differentiation, while bone dECM can enhance research, regenerative medicine, and tissue engineering.
osteogenic differentiation.
• Regulation of Stemness: The presence of specific ECM
components can maintain the balance between stem cell Discussion
self-renewal and differentiation, depending on the culture
conditions and the type of dECM used. The article’s examination of stem cell cultivation techniques is
3. Improved Cell Viability and Proliferation more than just a summary of recent developments; it’s a story that
• Reduced Apoptosis: The bioactive environment provided by spans decades of cellular biology’s evolutionary landscape and
dECM reduces cell stress and apoptosis, leading to higher captures decades of scientific research, invention, and hope. Stem
cell viability during culture. cells represent the unfathomable complexities of nature and provide
• Proliferative Support: The natural composition of dECM insight into the regeneration potential hidden in all living things.
can stimulate cell proliferation more effectively than They are frequently hailed as the cornerstone of regenerative
traditional culture substrates, allowing for the expansion medicine (Daley, 2012; Engle and Puppala, 2013).
of stem cell populations. The essay takes readers on a tour through the history of stem cell
4. Immunomodulatory Effects research, starting with the seminal findings of Takahashi and
• Immune Compatibility: dECM derived from autologous or Yamanaka, whose ground-breaking work revealed the
allogeneic cells can reduce the immune response in revolutionary idea of induced pluripotent stem cells (iPSCs).
regenerative medicine applications, which is critical for Because iPSCs can be derived from somatic cells and yet have
the survival and integration of transplanted stem cells. the pluripotency of embryonic stem cells, they have not only
• Anti-inflammatory Properties: Some dECM components changed the ethical landscape of stem cell research but have also
possess inherent anti-inflammatory properties, which can made cellular reprogramming more accessible to the public. This has
help in maintaining a conducive environment for stem cell opened new avenues for personalized medicine and disease
growth and function. modeling (Nonaka et al., 2004).
5. Customization and Versatility During the acknowledgment of these paradigm-shifting
• Source Flexibility: dECM can be derived from various discoveries, the piece also elucidates the complex interplay
tissues, enabling customization for specific stem cell between scientific advancement and inherent challenges. Stem
applications, such as neural, cardiac, or cell cultivation, with its intricate dynamics, presents significant
musculoskeletal tissues. obstacles for researchers. A primary concern is the genetic
• Combinatorial Approaches: dECM can be combined with instability observed in long-term cultures, which necessitates a
growth factors, cytokines, or other bioactive molecules to meticulous balance between preservation and expansion.
further tailor the microenvironment for specific stem Monitoring genetic stability is crucial to ensure that stem cells
cell needs. maintain their intended characteristics and avoid accumulating
6. Facilitating 3D Culture Systems deleterious mutations. Due to the inherent heterogeneity within
• 3D Scaffold Formation: dECM can be used to create three- stem cell populations—comparable to a painter’s palette—novel
dimensional (3D) culture systems that more accurately methodologies are required to achieve uniformity. Such
mimic the in vivo conditions, which is crucial for homogeneity is essential for effective therapeutic applications, as
studying cell behavior in a more realistic context. consistent genetic profiles are vital for reliable and successful
• Tissue Engineering: The use of dECM in 3D culture systems treatments (Ben-David, 2015; Briu et al., 2021; Turinetto
facilitates the development of complex tissue constructs, et al., 2017).
supporting the growth of stem cells in a manner that The essay lays out a path towards the horizon of possibility in
promotes tissue regeneration and repair. response to these difficulties, where cutting-edge innovations like
7. Reduction in Use of Animal-Derived Products machine learning and artificial intelligence (AI) serve as sentinels,
• Xeno-Free Cultures: dECM provides a viable alternative to pointing the way forward. AI integration offers a symbiotic
animal-derived matrices, such as Matrigel, in creating xeno- evolution where human creativity and data-driven insights
free culture environments, which is especially important for combine to create previously unimaginable levels of efficiency
clinical and translational applications. and reproducibility. This is in addition to optimization (Altyar
8. Potential for High-Throughput Screening et al., 2023; Nosrati et al., 2021).
• Scalability: dECM can be processed and applied in high- Furthermore, the conversation touches on a wide range of scientific
throughput screening platforms, allowing for the testing of topics, going beyond the boundaries of medicinal applications. Like the
various conditions and compounds on stem cells in a crucibles of alchemists, stem cell cultures are at the crossroads of several
controlled and reproducible manner. fields, ranging from ecological conservation to pulmonary biology. In
addition to providing a window into the secrets of disease pathology,
In summary, cell-derived dECM offers a multitude of benefits they hold the key to restoring the delicate balance of ecosystems,
for stem cell culture, including the provision of a native-like conserving endangered species, and opening the door to nature’s
microenvironment, enhanced differentiation and proliferation, resilience (Ponti et al., 2005; Todaro et al., 2007; Eramo et al., 2008;
and the ability to customize the scaffold for specific applications. Fischer et al., 2015).

Frontiers in Cell and Developmental Biology 08 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

However, amidst the grandeur of scientific endeavor, the article - Training, best practices, and cutting-edge technology are
grounds itself in the sobering reality of laboratory practice. It underscores imperative for progress in biomedical research.
the paramount importance of meticulous cell culture management,
where the pursuit of reliability and reproducibility stands as a Overall, the paper underscores the critical role of stem cell
sentinel against the specter of contamination and misidentification. culture in advancing medicine and emphasizes the importance of
Strategies ranging from stringent aseptic technique. The essay, addressing key challenges and leveraging emerging opportunities in
however, firmly roots itself in the grim realities of laboratory work, the field.
even amid the magnificence of scientific achievement. It emphasizes how
crucial it is to handle cell cultures meticulously, with the goal of
reproducibility and dependability acting as a guard against Author contributions
contamination and misidentification. Techniques varying from strict
aseptic methods to prudent use of antibiotics serve as the barrier against AG: Conceptualization, Investigation, Resources, Visualization,
the incursion of biological whims, guaranteeing that the foundation of Writing–original draft, Writing–review and editing. MT:
scientific investigation is integrity (Smith et al., 2014; Luizon et al., 2016). Conceptualization, Investigation, Project administration,
Finally, the paper becomes more than just a discussion; it becomes a Visualization, Writing–original draft, Writing–review and editing.
symphony that speaks to the goals, victories, and struggles that the BB: Conceptualization, Investigation, Visualization,
scientific community shares. It is evidence of the tenacious spirit of Writing–original draft, Writing–review and editing. AB:
inquiry, the unrelenting search for truth, and the unshakable faith in the Investigation, Resources, Visualization, Writing–original draft,
revolutionary potential of stem cell research to show the way towards a Writing–review and editing. SS: Funding acquisition,
more promising and health-conscious future for everybody. Writing–review and editing. RM: Formal Analysis, Investigation,
Project administration, Supervision, Visualization, Writing–original
draft, Writing–review and editing.
Conclusion
The paper explores advancements in stem cell research, covering Funding
methods, challenges, and perspectives. It discusses breakthroughs
like induced pluripotent stem cells (iPSCs) and hurdles such as The author(s) declare that no financial support was received for
genetic instability. It also highlights the potential of stem cell-derived the research, authorship, and/or publication of this article. This
products in personalized therapies. The section on stem cell origins research received no external funding.
includes perinatal tissues and induced pluripotent stem cells
(iPSCs), detailing acquisition methods and their benefits. It
emphasizes the importance of technology and molecular research Conflict of interest
in improving stem cell culture. The article discusses techniques for
generating iPSCs and human embryonic stem cells (hESCs) and The authors declare that the research was conducted in the
addresses common issues in cell culture management. absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
- iPSCs and hESCs present promising avenues for research
and therapy;
- Genetic instability poses a significant challenge in stem cell Publisher’s note
applications;
- Stem cell-derived products hold immense potential for All claims expressed in this article are solely those of the authors
personalized medicine; and do not necessarily represent those of their affiliated
- Perinatal tissues and iPSCs offer accessible sources for stem organizations, or those of the publisher, the editors and the
cell research; reviewers. Any product that may be evaluated in this article, or
- Technological advancements and molecular insights are claim that may be made by its manufacturer, is not guaranteed or
crucial for enhancing stem cell culture; endorsed by the publisher.

References
Aboul-Soud, M. A. M., Alzahrani, A. J., and Mahmoud, A. (2021). Induced Avar, M., Heinzer, D., Steinke, N., Dogancay, B., Moos, R., Lugan, S., et al. (2020).
pluripotent stem cells (iPSCs)-roles in regenerative therapies, disease modelling and Prion infection, transmission, and cytopathology modeled in a low-biohazard human
drug screening. Cells 10 (9), 2319. doi:10.3390/cells10092319 cell line. Life Sci. Alliance 3 (8), e202000814. doi:10.26508/lsa.202000814
Altyar, A. E., El-Sayed, A., Abdeen, A., Piscopo, M., Mousa, S. A., Najda, A., Babic, Z., Capes-Davis, A., Martone, M. E., Bairoch, A., Ozyurt, B., Gillespie, T. H.,
et al. (2023). Future regenerative medicine developments and their therapeutic et al. (2019). Incidences of problematic cell lines are lower in papers that use RRIDs to
applications. Biomed. & Pharmacother. 158, 114131. doi:10.1016/j.biopha.2022. identify cell lines. Elife e41676 8, e41676. doi:10.7554/eLife.41676
114131
Bacakova, L., Zarubova, J., Travnickova, M., Musilkova, J., Pajorova, J., Slepicka, P.,
American Type Culture Collection Standards Development Organization Workgroup et al. (2018). Stem cells: their source, potency and use in regenerative therapies with
ASN-0002 (2010). Cell line misidentification: the beginning of the end. Nat. Rev. Cancer focus on adipose-derived stem cells – a review. Biotechnol. Adv. 36 (4), 1111–1126.
10 (6), 441–448. doi:10.1038/nrc2852 doi:10.1016/j.biotechadv.2018.03.011

Frontiers in Cell and Developmental Biology 09 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

Baharvand, H., Hashemi, S. M., Kazemi Ashtiani, S., and Farrokhi, A. (2006). Eramo, A., Lotti, F., Sette, G., Pilozzi, E., Biffoni, M., Di Virgilio, A., et al. (2008).
Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D Identification and expansion of the tumorigenic lung cancer stem cell population. Cell
culture systems in vitro. Int. J. Dev. Biol. 50 (7), 645–652. doi:10.1387/ijdb.052072hb Death Differ. 15 (3), 504–514. doi:10.1038/sj.cdd.4402283
Barreto, S., Hamel, L., Schiatti, T., Yang, Y., and George, V. (2019). Cardiac progenitor Ferrúa, C. P., Centeno, E. G. Z., Rosa, L. C. D., Amaral, C. C. D., Severo, R. F.,
cells from stem cells: learning from genetics and biomaterials. Cells 8 (12), 1536. doi:10. Sarkis-Onofre, R., et al. (2017). How has dental pulp stem cells isolation been
3390/cells8121536 conducted? A scoping review. Braz Oral Res. 31, e87. doi:10.1590/1807-3107BOR-
2017.vol31.0087
Bartfeld, S., Bayram, T., van de Wetering, M., Huch, M., Begthel, H., Kujala, P., et al.
(2015). In vitro expansion of human gastric epithelial stem cells and their responses to Fischer, U., Backes, C., Raslan, A., Keller, A., Meier, C., and Meese, E. (2015). Gene
bacterial infection. Gastroenterology 148 (1), 126–136. doi:10.1053/j.gastro.2014.09.042 amplification during differentiation of mammalian neural stem cells in vitro and in vivo.
Oncotarget 6 (9), 7023–7039. doi:10.18632/oncotarget.3248
Ben-David, U. (2015). Genomic instability, driver genes and cell selection: projections
from cancer to stem cells. BBA Gene Regul. Mech. 1849 (4), 427–435. doi:10.1016/j. Gao, L., Xu, W., Li, T., Chen, J., Shao, A., Yan, F., et al. (2018). Stem cell therapy: a
bbagrm.2014.08.005 promising therapeutic method for intracerebral hemorrhage. Cell Transpl. 27 (12),
1809–1824. doi:10.1177/0963689718773363
Boeri, L., Albani, D., Raimondi, M. T., and Jacchetti, E. (2019). Mechanical regulation
of nucleocytoplasmic translocation in mesenchymal stem cells: characterization and Geraghty, R., Capes-Davis, A., Davis, J., Downward, J., Freshney, R. I., Knezevic, I.,
methods for investigation. Biophys. Rev. 11 (5), 817–831. doi:10.1007/s12551-019- et al. (2014). Guidelines for the use of cell lines in biomedical research. Br. J. Cancer 111
00594-3 (6), 1021–1046. doi:10.1038/bjc.2014.166
Borojevic, R., Monteiro, A. N., Vinhas, S. A., Domont, G. B., Mourao, P. A., Emonard, Giorgino, R., Albano, D., Fusco, S., Peretti, G. M., Mangiavini, L., and Messina, C.
H., et al. (1985). Establishment of a continuous cell line from fibrotic schistosomal (2023). Knee osteoarthritis: epidemiology, pathogenesis, and mesenchymal stem cells:
granulomas in mice livers. Vitro Cell Dev. Biol. 21 (7), 382–390. doi:10.1007/ what else is new? An update. Int. J. Mol. Sci. 24 (7), 6405. doi:10.3390/ijms24076405
BF02623469
Gombold, J., Karakasidis, S., Niksa, P., Podczasy, J., Neumann, K., Richardson, J., et al.
Briu, L. M., Maric, C., and Cadoret, J. C. (2021). Replication stress, genomic (2014). Systematic evaluation of in vitro and in vivo adventitious virus assays for the
instability, and replication timing: a complex relationship. Int. J. Mol. Sci. 22 (9), detection of viral contamination of cell banks and biological products. Vaccine 32 (24),
4764. doi:10.3390/ijms22094764 2916–2926. doi:10.1016/j.vaccine.2014.02.021
Cai, J., Chen, H., Xie, S., Hu, Z., and Bai, Y. (2022). Research progress of totipotent Grzelak, A., Rychlik, B., and Bartosz, G. (2001). Light-dependent generation of
stem cells. Stem Cells Dev. 31 (13-14), 335–345. doi:10.1089/scd.2022.0061 reactive oxygen species in cell culture media. Free Radic. Biol. Med. 30 (12),
1418–1425. doi:10.1016/s0891-5849(01)00545-7
Campostrini, G., Windt, L. M., van Meer, B. J., Bellin, M., and Mummery, C. L. (2021).
Cardiac tissues from stem cells: new routes to maturation and cardiac regeneration. Hendijani, F. (2017). Explant culture: an advantageous method for isolation of
Circ. Res. 128 (6), 775–801. doi:10.1161/CIRCRESAHA.121.318183 mesenchymal stem cells from human tissues. Cell Prolif. 50, e12334. doi:10.1111/
cpr.12334
Capes-Davis, A., Theodosopoulos, G., Atkin, I., Dexler, H. G., Kohara, A., MacLeod,
R. A. F., et al. (2010). Check your cultures! A list of cross-contaminated or misidentified Herbert, W. J., Parratt, D., Van Meirvenne, N., and Lennox, B. (1980). An accidental
cell lines. Int. J. Cancer 127 (1), 1–8. doi:10.1002/ijc.25242 laboratory infection with trypanosomes of a defined stock. II. Studies on the serological
response of the patient and the identity of the infecting organism. J. Infect. 2 (2),
Chen, F., Wang, H., and Xiao, J. (2020). Regulated differentiation of stem cells into an
113–124. doi:10.1016/s0163-4453(80)91109-3
artificial 3D liver as a transplantable source. Clin. Mol. Hepatol. 26 (2), 163–179. doi:10.
3350/cmh.2019.0022n Herwaldt, B. L. (2001). Laboratory-acquired parasitic infections from accidental
exposures. Clin. Microbiol. Rev. 14 (4), 659–688. doi:10.1128/CMR.14.3.659-688.2001
Chen, K. G., Mallon, B. S., McKay, R. D., and Robey, P. G. (2014). Human pluripotent
stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Huang, Y., Liu, Y., Zheng, C., and Shen, C. (2017). Investigation of cross-
Stem Cell 14 (1), 13–26. doi:10.1016/j.stem.2013.12.005 contamination and misidentification of 278 widely used tumor cell lines. PloS One
12 (1), e0170384. doi:10.1371/journal.pone.0170384
Chen, P., Zheng, L., Wang, Y., Tao, M., Xie, Z., Xia, C., et al. (2019). Desktop-
stereolithography 3D printing of a radially oriented extracellular matrix/mesenchymal Hwang, J. J., Rim, Y. A., Nam, Y., and Ju, J. H. (2021). Recent developments in clinical
stem cell exosome bioink for osteochondral defect regeneration. Theranostics 9 (9), applications of mesenchymal stem cells in the treatment of rheumatoid arthritis and
2439–2459. doi:10.7150/thno.31017 osteoarthritis. Front. Immunol. 12, 631291. doi:10.3389/fimmu.2021.631291
Cheng, H., Zheng, Z., and Cheng, T. (2020). New paradigms on hematopoietic stem Ilic, D., and Polak, J. M. (2011). Stem cells in regenerative medicine: introduction. Br.
cell differentiation. Protein Cell 11 (1), 34–44. doi:10.1007/s13238-019-0633-0 Med. Bull. 98, 117–126. doi:10.1093/bmb/ldr012
Chou, M. L., Bailey, A., Avory, T., Tanimoto, J., and Burnouf, T. (2015). Removal of Into, T., Kiura, K., Yasuda, M., Kataoka, H., Inoue, N., Hasebe, A., et al. (2004).
transmissible spongiform encephalopathy prion from large volumes of cell culture Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane
media supplemented with fetal bovine serum by using hollow fiber anion-exchange lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa
membrane chromatography. PLoS One 10 (4), e0122300. doi:10.1371/journal.pone. B activation. Cell Microbiol. 6 (2), 187–199. doi:10.1046/j.1462-5822.2003.00356.x
0122300
Jiang, Y., and Tuan, R. S. (2023). Bioactivity of human adult stem cells and functional
Christopher, L., and Baker, M. F. P. (2018). Capturing totipotent stem cells. Cell Stem relevance of stem cell-derived extracellular matrix in chondrogenesis. Stem Cell Res.
Cell 22 (1), 25–34. doi:10.1016/j.stem.2017.12.011 Ther. 14 (1), 160. doi:10.1186/s13287-023-03392-7
Chu, D. T., Phuong, T. N. T., Tien, N. L. B., Tran, D. K., Thanh, V. V., Quang, T. L., Karagiannis, P., Takahashi, K., Saito, M., Yoshida, Y., Okita, K., Watanabe, A., et al.
et al. (2020). An update on the progress of isolation, culture, storage, and clinical (2019). Induced pluripotent stem cells and their use in human models of disease and
application of human bone marrow mesenchymal stem/stromal cells. Int. J. Mol. Sci. 21 development. Physiol. Rev. 99 (1), 79–114. doi:10.1152/physrev.00039.2017
(3), 708. doi:10.3390/ijms21030708
Kim, Y. S., Majid, M., Melchiorri, A. J., and Mikos, A. G. (2018). Applications of
Daley, G. Q. (2012). The promise and perils of stem cell therapeutics. Cell Stem Cell 10 decellularized extracellular matrix in bone and cartilage tissue engineering. Bioeng. &
(6), 740–749. doi:10.1016/j.stem.2012.05.010 Transl. Med. 4 (1), 83–95. doi:10.1002/btm2.10110
de Morree, A., and Rando, T. A. (2023). Regulation of adult stem cell quiescence and Kobold, S., Bultjer, N., Stacey, G., Mueller, S. C., Kurtz, A., and Mah, N. (2023).
its functions in the maintenance of tissue integrity. Nat. Rev. Mol. Cell Biol. 24 (5), History and current status of clinical studies using human pluripotent stem cells. Stem
334–354. doi:10.1038/s41580-022-00568-6 Cell Rep. 18 (8), 1592–1598. doi:10.1016/j.stemcr.2023.03.005
Dierick, F., Solinc, J., Bignard, J., Soubrier, F., and Nadaud, S. (2021). Progenitor/stem Kolios, G., and Moodley, Y. (2013). Introduction to stem cells and regenerative
cells in vascular remodeling during pulmonary arterial hypertension. Cells 10 (6), 1338. medicine. Respiration 85 (1), 3–10. doi:10.1159/000345615
doi:10.3390/cells10061338
Lan, T., Luo, M., and Wei, X. (2021). Mesenchymal stem/stromal cells in cancer
Discher, D. E., Janmey, P., and Wang, Y. L. (2005). Tissue cells feel and respond to the therapy. J. Hematol. Oncol. 14 (1), 195. doi:10.1186/s13045-021-01208-w
stiffness of their substrate. Science 310 (5751), 1139–1143. doi:10.1126/science.1116995
Lee, J. Y., and Hong, S. H. (2020). Hematopoietic stem cells and their roles in tissue
Donnaloja, F., Jacchetti, E., Soncini, M., and Raimondi, M. T. (2020). Natural and regeneration. Int. J. Stem Cells 13 (1), 1–12. doi:10.15283/ijsc19127
synthetic polymers for bone scaffolds optimization. Polym. (Basel) 12 (4), 905. doi:10.
Lee, W. J., Lee, J. E., Hong, Y. J., Yoon, S. H., Song, H., Park, C., et al. (2020).
3390/polym12040905
Generation of brain organoids from mouse ESCs via teratoma formation. Stem Cell Res.
Drexler, H., and Uphoff, C. C. (2002). Mycoplasma contamination of cell cultures: 49, 102100. doi:10.1016/j.scr.2020.102100
incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39 (2),
Li, Y. R., Dunn, Z. S., Yu, Y., Li, M., Wang, P., and Yang, L. (2018). Advancing cell-
75–90. doi:10.1023/A:1022913015916
based cancer immunotherapy through stem cell engineering. Cell Stem Cell 30 (5),
Engle, S. J., and Puppala, D. (2013). Integrating human pluripotent stem cells into 592–610. doi:10.1016/j.stem.2023.02.009
drug development. Cell Stem Cell 12 (6), 669–677. doi:10.1016/j.stem.2013.05.011
Liu, G., David, B. T., Trawczynski, M., and Fessler, R. G. (2020). Advances in
Engler, A. J., Sen, S., Sweeney, H. L., and Discher, D. E. (2006). Matrix elasticity directs pluripotent stem cells: history, mechanisms, technologies, and applications. Stem Cell
stem cell lineage specification. Cell. 126 (4), 677–689. doi:10.1016/j.cell.2006.06.044 Rev. Rep. 16 (1), 3–32. doi:10.1007/s12015-019-09935-x

Frontiers in Cell and Developmental Biology 10 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

Llobet, L., Montoya, J., Lopez-Gallardo, E., and Ruiz-Pesini, E. (2015). Side effects of Ponti, D., Costa, A., Zaffaroni, N., Pratesi, G., Petrangolini, G., Coradini, D., et al.
culture media antibiotics on cell differentiation. Tissue Eng. Part C. Methods 21 (11), (2005). Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/
1143–1147. doi:10.1089/ten.TEC.2015.0062 progenitor cell properties. Cancer Res. 65 (13), 5506–5511. doi:10.1158/0008-5472.
CAN-05-0626
Lovell-Badge, R., Anthony, E., Barker, R. A., Bubela, T., Brivanlou, A. H., Carpenter,
M., et al. (2021). ISSCR guidelines for stem cell research and clinical translation: the Prieto Gonzalez, E. A. (2022). “A multilevel approach to the causes of genetic
2021 update. Stem Cell Rep. 16 (6), 1398–1408. doi:10.1016/j.stemcr.2021.05.012 instability in stem cells,” in Handbook of stem cell therapy. Springer, 1–55. doi:10.
1007/978-981-16-6016-0_26-1
Lu, V., Tennyson, M., Zhang, J., and Khan, W. (2021). Mesenchymal stem cell-
derived extracellular vesicles in tendon and ligament aepair: a systematic review of in Priya, N., Sarcar, S., Majumdar, A. S., and SundarRaj, S. (2014). Explant culture: a
vivo studies. Cells 10 (10), 2553. doi:10.3390/cells10102553 simple, reproducible, efficient, and economic technique for isolation of mesenchymal
stromal cells from human adipose tissue and lipoaspirate. J. Tissue Eng. Regen. Med. 8,
Luizon, M. R., Eckalbar, W. L., Wang, Y., Jones, S. L., Smith, P. R., Laurance, M., et al.
706–716. doi:10.1002/term.1569
(2016). Genomic characterization of metformin hepatic response. PLoS Genet. 12 (11),
e1006449. doi:10.1371/journal.pgen.1006449 Puerta-Alcalde, P., and Garcia-Vidal, C. (2021). Changing epidemiology of invasive
fungal disease in allogeneic hematopoietic stem cell transplantation. J. Fungi 7 (10), 848.
Maemura, M., Taketsuru, H., Nakajima, Y., Shao, R., Kakihara, A., Nogami, J., et al.
doi:10.3390/jof7100848
(2021). Totipotency of mouse zygotes extends to single blastomeres of embryos at the
four-cell stage. Sci. Rep. 11 (1), 11167. doi:10.1038/s41598-021-90653-1 Raitanen, J., Barta, B., Hacker, M., Georg, D., Balber, T., and Mitterhauser, M. (2023).
Comparison of radiation response between 2D and 3D cell culture models of different
Malekmohamadi, N., Abdanipour, A., Ghorbanlou, M., Shokri, S., Shirazi, R.,
human cancer cell lines. Cells 12 (3), 360. doi:10.3390/cells12030360
Dimitriadis, E., et al. (2019). Differentiation of bone marrow derived mesenchymal
stem cells into male germ-like cells in co-culture with testicular cells. Endocr. Regul. 53 Ratajczak, M. Z., Zuba-Surma, E., Kucia, M., Poniewierska, A., Suszynska, M., and
(2), 93–99. doi:10.2478/enr-2019-0011 Ratajczak, J. (2012). Pluripotent and multipotent stem cells in adult tissues. Adv. Med.
Sci. 57 (1), 1–17. doi:10.2478/v10039-012-0020-z
Malik, V., and Wang, J. (2022). Pursuing totipotency: authentic totipotent stem cells
in culture. Trends Genet. 38 (7), 632–636. doi:10.1016/j.tig.2022.03.012 Rey, F., Barzaghini, B., Nardini, A., Bordoni, M., Zuccotti, G. V., Cereda, C., et al.
(2020). Advances in tissue engineering and innovative fabrication techniques for 3-D-
Marega, M., El-Merhie, N., Gökyildirim, M. Y., Orth, V., Bellusci, S., and Chao, C. M.
structures: translational applications in neurodegenerative diseases. Cells 9 (7), 1636.
(2023). Stem/progenitor cells and related therapy in bronchopulmonary dysplasia. Int.
doi:10.3390/cells9071636
J. Mol. Sci. 24 (13), 11229. doi:10.3390/ijms241311229
Rodríguez-Fuentes, D. E., Fernández-Garza, L. E., Samia-Meza, J. A., Barrera-Barrera,
Mas-Bargues, C., and Borrás, C. (2021). Importance of stem cell culture conditions for
S. A., Caplan, A. I., and Barrera-Saldaña, H. A. (2021). Mesenchymal stem cells current
their derived extracellular vesicles therapeutic effect. Free Radic. Biol. Med. 168, 16–24.
clinical applications: a systematic review. Arch. Med. Res. 52 (1), 93–101. doi:10.1016/j.
doi:10.1016/j.freeradbiomed.2021.03.028
arcmed.2020.08.006
McKee, C., and Chaudhry, G. R. (2017). Advances, and challenges in stem cell culture.
Rottem, S. (2003). Interaction of mycoplasmas with host cells. Physiol. Rev. 83 (2),
Colloids Surf. B Biointerfaces 159, 62–77. doi:10.1016/j.colsurfb.2017.07.051
417–432. doi:10.1152/physrev.00030.2002
Meten, O. W. (2002). Virus contaminations of cell cultures–a biotechnological view.
Ryu, A. H., Eckalbar, W. L., Kreimer, A., Yosef, N., and Ahituv, N. (2017). Use
Cytotechnology 39, 91–116. doi:10.1023/A:1022969101804
antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced
Mushahary, D., Spittler, A., Kasper, C., Weber, V., and Charwat, V. (2018). Isolation, changes in gene expression and regulation. Sci. Rep. 7 (1), 7533. doi:10.1038/s41598-
cultivation, and characterization of human mesenchymal stem cells. Cytometry 93, 017-07757-w
19–31. doi:10.1002/cyto.a.23242
Sajeesh, S., Broekelman, T., Mecham, R. P., and Ramamurthi, A. (2020). Stem cell
Nava, M. M., Raimondi, M. T., and Pietrabissa, R. (2012). Controlling self-renewal derived extracellular vesicles for vascular elastic matrix regenerative repair. Acta
and differentiation of stem cells via mechanical cues. J. Biomed. Biotechnol. 2012, Biomater. 113, 267–278. doi:10.1016/j.actbio.2020.07.002
797410. doi:10.1155/2012/797410
Sanders, R. C., Slayton, W. B., Cogle, C. R., Fisher, R. C., and Scott, E. W. (2006). Stem
Nelson-Rees, W. A., and Flandermeyer, R. R. (1976). HeLa cultures defined. Science cell research. Pediatr. Respir. Rev. 7 (2), 135–140. doi:10.1016/j.prrv.2006.03.009
191 (4222), 96–98. doi:10.1126/science.1246601
Schroder, S. K., Schuler, H. M., Petersen, K. V., Tesauro, C., Knudsen, B. R.,
Nikitin, P. V., Musina, G. R., Polozov, V. N., Goreiko, D. N., Krasnovsky, V. M., Pedersen, F. S., et al. (2022). Genetic and molecular characterization of the
Werkenbark, L., et al. (2023). Development of glioblastoma from stem cells to a full- immortalized murine hepatic stellate cell line GRX. Cells 11 (9), 1504. doi:10.
fledged tumor. Turk Patoloji Derg. 39 (2), 117–132. doi:10.5146/tjpath.2020.01582 3390/cells11091504
Nims, R. W., and Price, P. J. (2017). Best practices for detecting and mitigating the risk Slack, J. (2018). What is a stem cell? Wiley Interdiscip. Rev. Dev. Biol. 7 (5), e323.
of cell culture contaminants. Vitro Cell. & Dev. Biology-Animal 53 (10), 872–879. doi:10. doi:10.1002/wdev.323
1007/s11626-017-0203-9
Smith, R. P., Eckalbar, W. L., Morrissey, K. M., Luizon, M. R., Hoffman, T. J., Sun, X.,
Nonaka, M., Yoshikawa, M., Nishimura, F., Yokota, H., Kimura, H., Hirabayashi, H., et al. (2014). Genome-wide discovery of drug-dependent human liver regulatory
et al. (2004). Intraventricular transplantation of embryonic stem cell-derived neural elements. PloS Genet. 10 (10), e1004648. doi:10.1371/journal.pgen.1004648
stem cells in intracerebral hemorrhage rats. Neurol. Res. 26 (3), 265–272. doi:10.1179/
Sobhani, A., Khanlarkhani, N., Baazm, M., Mohammadzadeh, F., Najafi, A.,
016164104225014049
Mehdinejadiani, S., et al. (2017). Multipotent stem cell and current application. Acta
Nosrati, H., Khouy, R. A., Nosrati, A., Khodaei, M., Banitalebi-Dehkordi, M., Ashrafi- Med. Iran. 55 (1), 6–23.
Dehkordi, K., et al. (2021). Nanocomposite scaffolds for accelerating chronic wound
Souza, A. G., Silva, I. B. B., Campos-Fernandez, E., Barcelos, L. S., Souza, J. B.,
healing by enhancing angiogenesis. J. Nanobiotechnology 19 (1), 1. doi:10.1186/s12951-
Marangoni, K., et al. (2018). Comparative assay of 2D and 3D cell culture models:
020-00755-7
proliferation, gene expression and anticancer drug response. Curr. Pharm. Des. 24 (15),
Nosrati, H., and Nosrati, M. (2023). Artificial intelligence in regenerative medicine: 1689–1694. doi:10.2174/1381612824666180404152304
applications and implications. Biomimetics 8 (5), 442. doi:10.3390/biomimetics8050442
Steimberg, N., Bertero, A., Chiono, V., Dell’Era, P., Di Angelantonio, S., Hartung, T.,
Orticelli, V., Papait, A., Vertua, E., Bonassi Signoroni, P., Romele, P., Di Pietro, L., et al. (2020). iPS, organoids and 3D models as advanced tools for in vitro toxicology.
et al. (2021). Human amniotic mesenchymal stromal cells support the ex vivo expansion ALTEX 37 (1), 136–140. doi:10.14573/altex.1911071
of cord blood hematopoietic stem cells. Stem Cells Transl. Med. 10 (11), 1516–1529.
Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from
doi:10.1002/sctm.21-0130
mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 (4), 663–676.
Otto, W. R., and Green, A. M. (2020). Fungal infections in children with haematologic doi:10.1016/j.cell.2006.07.024
malignancies and stem cell transplant recipients. Br. J. Hematol. 189 (4), 607–624.
Takahashi, K., and Yamanaka, S. (2013). Induced pluripotent stem cells in medicine
doi:10.1111/bjh.16452
and biology. Development 140 (12), 2457–2461. doi:10.1242/dev.092551
Palumbo, P., Lombardi, F., Siragusa, G., Cifone, M. G., Cinque, B., and Giuliani, M. (2018).
Taur, Y., Jenq, R. R., Parales, M. A., Littmann, E. R., Morjaria, S., Ling, L., et al. (2014).
Methods of isolation, characterization, and expansion of human adipose-derived stem cells
The effects of intestinal tract bacterial diversity on mortality following allogenic
(ASCs): an overview. Int. J. Mol. Sci. 19 (7), 1897. doi:10.3390/ijms19071897
hematopoietic steam cell transplantation. Blood 124 (7), 1174–1182. doi:10.1182/
Pamies, D., Leist, M., Coecke, S., Bowe, G., Allen, D. G., Gstraunthaler, G., et al. blood-2014-02-554725
(2022). Guidance document on good cell and tissue culture practice 2.0 (GCCP 2.0).
Todaro, M., Alea, M. P., Di Stefano, A. D., Cammareri, P., Vermeulen, L., Iovino, F.,
Altern. Animal Exp. 39 (1), 30–70. doi:10.14573/altex.2111011
et al. (2007). Colon cancer stem cells dictate tumor growth and resist cell death by
Parrotta, E. I., Scalise, S., Scaramuzzino, L., and Cuda, G. (2019). Stem cells: the game production of interleukin 4. Cell Stem Cell 1 (4), 389–402. doi:10.1016/j.stem.2007.
changers of human cardiac disease modelling and regenerative medicine. Int. J. Mol. Sci. 08.001
20 (22), 5760. doi:10.3390/ijms20225760
Todtenhaupt, P., Franken, L. A., Groene, S. G., van Hoolwerff, M., van der Meeren, L.
Pei, M. (2017). Environmental preconditioning rejuvenates adult stem cells’ E., van Klink, J. M. M., et al. (2023). A robust and standardized method to isolate and
proliferation and chondrogenic potential. Biomaterials 117, 10–23. doi:10.1016/j. expand mesenchymal stromal cells from human umbilical cord. Cytotherapy 25 (10),
biomaterials.2016.11.049 1057–1068. doi:10.1016/j.jcyt.2023.07.004

Frontiers in Cell and Developmental Biology 11 frontiersin.org

Górska et al. 10.3389/fcell.2024.1435461

Turinetto, V., Orlando, L., and Giachino, C. (2017). Induced pluripotent stem cells: Yen, B. L., Hsieh, C. C., Hsu, P. J., Chang, C. C., Wang, L. T., and Yen, M. L. (2023).
advances in the quest for genetic stability during reprogramming process. Int. J. Mol. Sci. Three-dimensional spheroid culture of human mesenchymal stem cells: offering
18 (9), 1952. doi:10.3390/ijms18091952 therapeutic advantages and in vitro glimpses of the in vivo state. Stem Cells Transl.
Med. 12 (5), 235–244. doi:10.1093/stcltm/szad011
Varghese, D. S., Paeween, S., Ardah, M. T., Starling Emerald, B., and Ansari, S. A.
(2017). Effects of aminoglycoside antibiotics on human embryonic stem cell viability Ylostalo, J. H. (2020). 3D stem cell culture. Cells 9 (10), 2178. doi:10.3390/
during differentiation in vitro. Stem Cells Int. 2017, 2451927. doi:10.1155/2017/2451927 cells9102178
Wang, Y., Hu, G., Hill, R. C., Dzieciatkowska, M., Hansen, K. C., Zhang, X. B., et al. Young, R. A. (2011). Control of the embryonic stem cell state. Cell 144 (6), 940–954.
(2021a). Matrix reverses immortalization-mediated stem cell fate determination. doi:10.1016/j.cell.2011.01.032
Biomaterials 265, 120387. doi:10.1016/j.biomaterials.2020.120387
Yu, N., Zhang, F., Tang, X., Liu, Y., Zhang, J., Yang, B., et al. (2023). Hierarchical
Wang, Y. H., Tao, Y. C., Wu, D. B., Wang, M. L., Tang, H., and Chen, E. Q. (2021b). hydrogel microarrays fabricated based on a microfluidic printing platform for high-
Cell heterogeneity, rather than the cell storage solution, affects the behavior of throughput screening of stem cell lineage specification. Acta Biomater. 161, 144–153.
mesenchymal stem cells in vitro and in vivo. Stem Cell Res. Ther. 12, 391. doi:10. doi:10.1016/j.actbio.2023.02.036
1186/s13287-021-02450-2
Zakrzewski, W., Dobrzyński, M., Szymonowicz, M., and Rybak, Z. (2019). Stem cells:
Ware, C. B., Jonlin, E. C., Anderson, D. J., Cavanaugh, C., Hesson, J., Sidhu, S., et al. past, present, and future. Stem Cell Res. Ther. 10 (1), 68. doi:10.1186/s13287-019-1165-5
(2023). Derivation of naïve human embryonic stem cells using a CHK1 inhibitor. Stem
Zaripova, L. N., Midgley, A., Christmas, S. E., Beresford, M. W., Pain, C., Baildam, E.
Cell Rev. Rep. 19 (8), 2980–2990. doi:10.1007/s12015-023-10613-2
M., et al. (2023). Mesenchymal stem cells in the pathogenesis and therapy of
Weiskirchen, R. (2022). Established liver cell lines: are you sure to have the right ones? autoimmune and autoinflammatory diseases. Int. J. Mol. Sci. 24 (22), 16040. doi:10.
Livers 2 (3), 171–177. doi:10.3390/livers2030015 3390/ijms242216040
Wilkinson, A. C., Ishida, R., Nakauchi, H., and Yamazaki, S. (2020). Long-term ex Zeyu, T., Tao, Y., Jun, L., Ting, W., and Akon, H. (2023). Introduction to stem cells.
vivo expansion of mouse hematopoietic stem cells. Nat. Protoc. 15 (2), 628–648. doi:10. Prog. Mol. Biol. Transl. Sci. 199, 3–32. doi:10.1016/bs.pmbts.2023.02.012
1038/s41596-019-0263-2
Zhang, X., Chen, X., Hong, H., Hu, R., Liu, J., and Liu, C. (2021). Decellularized
Xie, X., Nóbrega, R., and Pšenička, M. (2020). Spermatogonial stem cells in fish: extracellular matrix scaffolds: recent trends and emerging strategies in tissue
characterization, isolation, enrichment, and recent advances of in vitro culture systems. engineering. Bioact. Mater. 10, 15–31. doi:10.1016/j.bioactmat.2021.09.014
Biomolecules 10 (4), 644. doi:10.3390/biom10040644
Zhao, Y., Wang, Q., Zhang, W., Jia, Q., Gao, Q., and Shuai, L. (2023). Protocol to
Yamanaka, S. (2020). Pluripotent stem cell-based cell therapy-promise and generate induced trophoblast stem cells from embryonic stem cells in mice. Star. Protoc.
challenges. Cell Stem Cell 27 (4), 523–531. doi:10.1016/j.stem.2020.09.014 4 (1), 102092. doi:10.1016/j.xpro.2023.102092
Yao, Q., Zheng, Y. W., Lan, Q. H., Kou, L., Xu, H. L., and Zhao, Y. Z. (2019). Recent Zigler, J. S., Lepe-Zuniga, J. L., Vistirca, B., and Gery, I. (1985). Analysis of the
development and biomedical applications of decellularized extracellular matrix biomaterials. cytotoxic effects of light-exposed HEPES-containing culture medium. Vitro Cell. & Dev.
Mater. Sci. & Eng. C, Mater. Biol. Appl. 104, 109942. doi:10.1016/j.msec.2019.109942 Biol. 21 (5), 282–287. doi:10.1007/BF02620943

Frontiers in Cell and Developmental Biology 12 frontiersin.org