1.
Animal Cell Culture Laboratory: Standard Practices and Sterile Techniques
Aim:
To establish and maintain a sterile environment for culturing animal cells by following standard
laboratory practices and aseptic techniques, ensuring contamination-free cell growth and reproducible
experimental results.
Principle:
Animal cell culture involves the in vitro maintenance and propagation of cells in a controlled
environment using nutrient-rich culture media. Cells are highly susceptible to contamination from
bacteria, fungi, and viruses; thus, strict sterile techniques and aseptic handling are essential. The key
principles include:
Maintaining sterility through proper disinfection and laminar airflow usage.
Providing optimal growth conditions, including temperature, CO₂ levels, humidity, and
nutrients.
Preventing cross-contamination by using dedicated equipment and handling techniques.
Procedure:
1. Preparation of the Laboratory & Equipment:
o Disinfect all surfaces with 70% ethanol.
o Sterilize culture media, reagents, and glassware using autoclaving or filtration.
o Maintain a sterile workspace using a laminar flow hood.
2. Aseptic Handling Techniques:
o Wear gloves, lab coats, and masks.
o Spray hands and materials with ethanol before placing them inside the laminar hood.
o Work near the flame or in the sterile airflow to minimize contamination.
3. Cell Culture Initiation & Maintenance:
o Thaw frozen cells quickly in a 37°C water bath and transfer them to pre-warmed
media.
o Centrifuge cells at low speed and resuspend them in fresh media.
o Incubate cells at 37°C with 5% CO₂.
4. Passaging (Subculturing) Cells:
o Remove old media and wash cells with PBS.
o Add trypsin-EDTA to detach adherent cells.
o Neutralize trypsin with complete media and transfer cells to new flasks.
5. Contamination Control & Monitoring:
o Regularly check cultures under a microscope for microbial contamination.
o Discard contaminated cultures immediately.
o Use antibiotics only when necessary to avoid masking contamination.
Results:
Through this experiment, we learned the importance of maintaining aseptic conditions in an animal
cell culture laboratory. Proper disinfection, sterile handling, and contamination control ensure
healthy cell growth. The use of laminar flow hoods, personal protective equipment (PPE), and proper
waste disposal minimizes microbial contamination. We also understood that maintaining optimal
environmental conditions (temperature, CO₂, humidity) is crucial for successful cell culture.
2. Reagents and Media Preparation for Animal Cell Culture
Aim:
To prepare and sterilize culture media and essential reagents required for maintaining and propagating
animal cells under sterile conditions.
Principle:
Animal cell culture requires a nutrient-rich medium that provides essential amino acids, vitamins,
glucose, salts, and growth factors to support cell survival and proliferation. The medium's pH,
osmolality, and sterility must be carefully maintained. Commonly used culture media include DMEM,
RPMI-1640, and MEM, supplemented with fetal bovine serum (FBS), antibiotics, and buffering
agents. Filtration or autoclaving ensures sterility before use.
Procedure:
1. Preparation of Culture Media:
o Weigh and dissolve the powdered medium (e.g., DMEM, RPMI) in deionized water.
o Adjust the pH to ~7.2–7.4 using NaOH or HCl.
o Add required supplements such as:
10% Fetal Bovine Serum (FBS)
1% Penicillin-Streptomycin (antibiotics)
2 mM L-glutamine
o Filter sterilize the medium using a 0.22 µm membrane filter under aseptic conditions.
o Store the prepared media at 4°C until use.
2. Preparation of PBS (Phosphate-Buffered Saline):
o Dissolve NaCl, KCl, Na₂HPO₄, and KH₂PO₄ in deionized water.
o Adjust the pH to 7.2–7.4.
o Sterilize by autoclaving or filtration.
3. Preparation of Trypsin-EDTA Solution:
o Dissolve trypsin and EDTA in PBS.
o Filter sterilize using a 0.22 µm membrane.
o Aliquot and store at -20°C or 4°C.
4. Sterility Check:
o Incubate a small aliquot of the prepared media and reagents at 37°C for 24–48 hours
to confirm sterility before use.
Results:
From this experiment, we learned that preparing and sterilizing culture media is fundamental for
successful cell growth. Proper pH adjustment, filtration, and storage techniques prevent
contamination. The role of fetal bovine serum (FBS), antibiotics, and buffering agents in cell culture
media was also understood. Additionally, we learned that sterility testing before use ensures reliable
and reproducible results in experiments.
3. Subculturing of Human Embryonic Kidney (HEK) Cells
Aim:
To passage (subculture) Human Embryonic Kidney (HEK) cells to maintain continuous growth,
prevent overconfluency, and sustain their viability for experimental use.
Principle:
HEK cells, commonly grown as adherent cultures, require regular subculturing to prevent overgrowth
and maintain healthy proliferation. Subculturing involves detaching the cells from the culture surface
using trypsin-EDTA, resuspending them in fresh medium, and transferring them to new culture flasks.
Aseptic techniques are essential to avoid contamination.
Procedure:
1. Preparation:
o Warm the complete culture medium (e.g., DMEM with 10% FBS) and trypsin-EDTA
to 37°C.
o Label new culture flasks and place all materials inside a laminar flow hood.
2. Cell Observation & Media Removal:
o Examine the HEK cells under an inverted microscope to check for confluency
(typically 70–80% for subculturing).
o Aspirate and discard the old culture medium carefully.
3. Cell Detachment:
o Rinse the cells gently with sterile PBS to remove residual serum.
o Add 1–2 mL of trypsin-EDTA to the flask and incubate at 37°C for 2–5 minutes until
cells detach (observe under a microscope).
o Tap the flask gently if necessary to aid detachment.
4. Neutralization & Resuspension:
o Add an equal volume of complete medium to inactivate trypsin.
o Pipette the cell suspension gently to break clumps and ensure even distribution.
5. Cell Counting (Optional):
o Take an aliquot of the suspension and mix with trypan blue.
o Count viable cells using a hemocytometer or an automated cell counter.
6. Seeding New Culture Flasks:
o Transfer an appropriate volume of the cell suspension into new flasks with fresh pre-
warmed medium.
o Maintain a suitable seeding density (e.g., 1:3 or 1:5 dilution).
7. Incubation:
o Place flasks in a 37°C incubator with 5% CO₂.
o Allow cells to attach and grow for the next 24–48 hours before further observation.
Results:
We learned that subculturing is necessary to maintain HEK cells and prevent overgrowth. The
process involves detaching cells with trypsin-EDTA, resuspending them in fresh media, and
transferring them to new culture flasks. Proper aseptic handling ensures high cell viability and
prevents contamination. We also understood that cell density, passage number, and media changes
impact the overall health and growth of the cells.
4. Cryopreservation of Human Embryonic Kidney (HEK) Cells
Aim:
To preserve Human Embryonic Kidney (HEK) cells at ultra-low temperatures for long-term storage
while maintaining their viability and functionality upon thawing.
Principle:
Cryopreservation involves freezing cells at -80°C or in liquid nitrogen (-196°C) using a
cryoprotective agent (e.g., DMSO) to prevent ice crystal formation, which can damage cell
membranes. A slow freezing process ensures minimal stress, and proper storage conditions help
maintain cell integrity for future use.
Procedure:
1. Preparation:
o Warm the complete culture medium and prepare freezing medium (90% FBS + 10%
DMSO).
o Label cryovials with the cell line name, passage number, and date.
2. Harvesting Cells:
o Check cell confluency (70–80%) under a microscope.
o Aspirate old medium and wash cells with PBS.
o Add trypsin-EDTA and incubate at 37°C until cells detach (2–5 minutes).
o Neutralize trypsin with complete medium and transfer the suspension to a centrifuge
tube.
3. Centrifugation & Resuspension:
o Centrifuge at 1,000 rpm for 5 minutes.
o Discard the supernatant and gently resuspend the pellet in cold freezing medium.
4. Aliquoting & Freezing:
o Dispense 1 mL of the cell suspension into sterile cryovials.
o Place cryovials in a controlled-rate freezing container (e.g., Mr. Frosty) and store at -
80°C overnight for slow freezing.
5. Liquid Nitrogen Storage:
o After 24 hours, transfer the cryovials to liquid nitrogen (-196°C) for long-term
storage.
Results:
We learned that cryopreservation allows long-term storage of HEK cells without losing their viability.
The use of cryoprotectants like DMSO prevents ice crystal formation. A slow freezing process at -
80°C, followed by storage in liquid nitrogen, ensures maximum cell survival. We also understood that
improper freezing or thawing techniques can result in high cell death rates.
5. Revival of Human Embryonic Kidney (HEK) Cells
Aim:
To revive cryopreserved Human Embryonic Kidney (HEK) cells from liquid nitrogen storage and
restore their normal growth and proliferation in culture conditions.
Principle:
Thawing frozen HEK cells must be done rapidly to prevent ice crystal formation, which can damage
cell membranes. Once thawed, cells are immediately transferred to pre-warmed culture medium to
dilute out the cryoprotectant (DMSO) and promote cell recovery. Proper handling and sterile
techniques ensure high cell viability and minimal stress.
Procedure:
1. Preparation:
o Warm complete culture medium (e.g., DMEM with 10% FBS) to 37°C.
o Pre-warm the incubator (37°C, 5% CO₂).
o Label culture flasks and prepare a laminar flow hood for aseptic handling.
2. Thawing the Cells:
o Retrieve the cryovial from liquid nitrogen and immediately place it in a 37°C water
bath.
o Swirl gently and thaw rapidly (~1–2 minutes) until only a small ice pellet remains.
o Wipe the cryovial with 70% ethanol before opening inside a laminar flow hood.
3. Dilution & Centrifugation:
o Transfer the thawed cell suspension into a 15 mL tube containing 10 mL of pre-
warmed complete medium.
o Centrifuge at 1,000 rpm for 5 minutes to remove DMSO.
o Carefully aspirate the supernatant and resuspend the pellet in fresh medium.
4. Seeding into Culture Flask:
o Transfer the resuspended cells to a new culture flask containing pre-warmed medium.
o Incubate at 37°C with 5% CO₂.
5. Post-Thaw Monitoring:
o After 24 hours, check cell attachment and morphology under a microscope.
o Replace the medium to remove any residual dead cells.
o Continue regular maintenance and subculturing as needed.
Results:
We understood that the revival process requires rapid thawing in a 37°C water bath to prevent
damage. Dilution with fresh media and removal of DMSO through centrifugation help restore normal
cell function. Proper incubation conditions allow cells to adhere and recover within 24–48 hours. We
also learned that careful monitoring is essential to ensure that revived cells regain normal
morphology and proliferative ability.
6. Animal House: Standard Practices and Sterile Techniques
Aim:
To establish and maintain a controlled and hygienic environment in an animal house to ensure the
well-being of laboratory animals, prevent infections, and ensure reliable experimental outcomes.
Principle:
The animal house provides a controlled environment for the housing, breeding, and experimentation
of laboratory animals under ethical and sterile conditions. Proper maintenance, aseptic handling, and
biosecurity measures help prevent disease transmission, stress, and contamination, ensuring animal
welfare and research integrity.
Procedure:
1. Animal House Facility Maintenance:
o Maintain controlled temperature (20–24°C), humidity (40–60%), and ventilation.
o Follow a 12-hour light/dark cycle to maintain animal circadian rhythms.
o Use appropriate caging systems based on species (e.g., individually ventilated cages
for rodents).
2. Sterile Techniques & Biosecurity Measures:
o Restrict entry to authorized personnel only.
o Wear sterile lab coats, gloves, shoe covers, and masks before handling animals.
o Disinfect work areas, cages, and feeding equipment regularly.
o Autoclave bedding, feed, and water before use.
3. Animal Handling & Care:
o Handle animals gently using species-specific techniques to minimize stress.
o Quarantine new arrivals for health screening before introducing them to the colony.
o Monitor for signs of illness, injury, or abnormal behavior.
4. Feeding & Watering:
o Provide species-appropriate, nutritionally balanced feed.
o Use sterilized water to prevent microbial contamination.
o Clean and replace food and water containers daily.
5. Waste Management & Hygiene:
o Dispose of animal waste and used bedding in biohazard containers.
o Regularly sanitize cages and change bedding to prevent infections.
o Implement proper pest control measures.
6. Experimental Procedures & Ethical Considerations:
o Follow Institutional Animal Ethics Committee (IAEC) guidelines.
o Use anesthesia and analgesics as required to minimize pain.
o Euthanize animals humanely if necessary, using approved methods.
Results:
We learned that an animal house must maintain strict hygiene and environmental conditions to
ensure the well-being of laboratory animals. Sterile handling, appropriate feeding, and ethical
practices prevent infections and stress. The importance of quarantine, waste management, and
biosecurity measures in preventing disease transmission was also understood. Additionally, we
learned that proper experimental procedures, as per ethical guidelines, help obtain reliable research
outcomes while ensuring animal welfare.
