Totipotent stem cell culture

Totipotent stem cells refer to all cells from the fertilized egg up to the 32-cell cleavage stage; they are capable of differentiating and developing into various tissues and organs. They possess high totipotency, with the potential for unlimited differentiation and the ability to form a complete organism. Some facts about human pluripotent stem cells (PSCs): 1. They attach to several types of extracellular matrix (ECM), including Matrigel, laminin, vitronectin, fibronectin, and high-density RGD peptides. The best and most natural ECM for human PSCs is laminin-511 or -521. 2. Human PSCs do not thrive as single cells; they consistently grow best in colonies. 3. The doubling time for human PSCs in E8 medium is 16–20 hours. 4. Cells typically divide every 4–5 days. 5. A single well of a 6-well plate at 90% confluence yields approximately 1.5–2 million cells.

MCE has not independently verified the accuracy of these methods. They are provided for reference only.

DMEM/F12 (GIBCO, 11330032) and Neurobasal (GIBCO, 21103049), 0.5X N2 supplement (GIBCO, 17502048), 0.5X B27 supplement (GIBCO, 17504044), 2% BSA, 1X L-glutamine, 1X penicillin-streptomycin, 50 mM 2-mercaptoethanol, 1000 U/mL mLIF, 3 mM CHIR99021 (Selleck, S1263) and 0.5 mM PD0325901 (Selleck, S1036), SLP medium (serum/LIF medium supplemented with 2.5 nM PlaB (Tocris, 6070)), 0.25% trypsin-EDTA.

1. Temporary Storage Requirements
Remove the target cryovial from the liquid nitrogen tank. If subsequent steps must be paused, place the cryovial on dry ice for temporary storage to prevent passive thawing at room temperature.
2. Rapid Thawing
Place the cryovial in a 37°C water bath for rapid thawing, gently agitating it to accelerate the process. Use a floating rack to secure the cryovial so that approximately two-thirds of the vial is submerged and one-third remains above the water line; this ensures adequate heating while preventing contamination from water bath backflow.
Remove the vial immediately once most of the contents have thawed (leaving only a small amount of ice crystals); do not continue heating after complete thawing, in order to minimize DMSO-induced toxicity.
Safety Warning: Defective cryovials may burst due to a sudden rise in internal pressure during thawing. Safety goggles must be worn throughout the procedure, and the face should not be positioned directly over the water bath.
3. Disinfection and Transfer
Spray the exterior of the cryovial with 70% ethanol; allow it to dry before transferring it to a biosafety cabinet (or fume hood) to open the cap. Use a P1000 pipette to gently transfer the cell suspension into a 15 mL conical centrifuge tube.
4. Gradual Dilution with Culture Medium (to reduce osmotic shock)
Slowly add 5–10 mL of fresh E8 medium to the centrifuge tube, gently mixing as you add. The first milliliter must be added dropwise to allow the cells to gradually adapt to osmotic changes, preventing damage caused by sudden exposure to a hypertonic environment.
Do not pour the thawed cell suspension directly into a large volume of fresh medium.
5. Centrifugation and Resuspension
Place the centrifuge tube in the centrifuge and spin at 150 × g for 3 minutes. Carefully aspirate and discard the supernatant, then add an appropriate amount of fresh E8..." ...culture medium, and gently pipette up and down to resuspend the cell pellet.
6. Seeding
Seed the cell suspension into a culture plate pre-coated with Matrigel; gently swirl to distribute evenly, then incubate undisturbed at 37°C in a 5% CO₂ incubator.
7. Observation of cell attachment
Cells should complete attachment within approximately 1 hour after seeding.
8. Next-day inspection and viability assessment
Examine the cell status under a microscope the day after thawing. The presence of a small number of floating dead cells is normal. Cell viability typically ranges from 40% to 70%, depending on the quality of cryopreservation and the thawing procedure.

Routine medium change: Replace with fresh E8 medium once daily; discard the old medium and add an equal volume of fresh E8 (pre-warmed or at room temperature).
Weekend / Low-density double-volume medium change: When cell confluence is low (e.g., shortly after passaging), a double volume of medium may be used; this allows for skipping medium changes for several days, making it suitable for maintenance during unattended periods such as weekends.

Note: After passaging, incubate the cells at 37°C in a 5% CO₂ incubator.

When cell confluence reaches 70%-90%, subculture should be performed promptly to prevent differentiation or apoptosis caused by over-confluence.

1. Subculture procedure (using a single well of a 6-well plate as an example)
The volumes specified below are for a single well of a 6-well plate; when using other culture vessels, adjust solution volumes proportionally based on the surface area.
1: Rinsing prior to EDTA dissociation
Aspirate and discard the spent medium from the well. Add 1 mL of PBS + EDTA (final concentration 0.5 mM), gently rinse the cell layer, and aspirate the rinse solution.
2: EDTA incubation for dissociation
Add another 1 mL of PBS + EDTA (0.5 mM) and place the culture plate in a biosafety cabinet at room temperature for 3-5 minutes to allow EDTA to chelate calcium ions and disrupt intercellular junctions.
3: Microscopic verification of dissociation status
Observe under an inverted microscope: cells within colonies should begin to show signs of separation (widening intercellular gaps, retraction of colony edges). If cells remain tightly adherent, incubate for an additional 2-3 minutes before re-examining; avoid over-dissociation.
4: Aspirate EDTA
Carefully aspirate the PBS + EDTA solution from the well.
Note: If the EDTA incubation time is excessive, cells may detach from the culture surface during this step and be lost along with the aspirated liquid. Exercise extreme caution to avoid aspirating the target cells.
5: Resuspension in medium and dissociation of cell clumps
Using a 5 mL pipette, add 3 mL of fresh E8 medium. Detach cells from the surface solely by the force of the medium flow; do not scrape cells with pipette tips or cell scrapers. Gently pipette the cell suspension up and down several times to break up cell clumps into appropriately sized clusters (hPSC"" (Passaging typically retains small cell clusters rather than single cells.)
6: Seeding
Aliquot the cell suspension according to the desired dilution ratio and seed into new culture plates pre-filled with fresh E8 medium; gently rock the plates in a crosswise motion to distribute the cells evenly, then incubate.

Dilution Ratio	Time to Reach Target Density	Target Confluency	Application
1:6	Approx. 2 days	50%-70%	Suitable for cell densities required for experiments such as electroporation
1:12	Approx. 3 days	70%-80%	Routine subculture and maintenance
1:20	Approx. 4-5 days	70%-90%	Routine maintenance; culture with double-volume medium change over the weekend

Preparation Before Cryopreservation
1. Optimizing Cell Status
Subculture the cells at a 1:3 or 1:6 ratio two days prior to cryopreservation. Cells are in the logarithmic growth phase on the second day after subculturing, yielding the highest post-thaw viability.
If experimental scheduling is constrained, cells harvested 4-5 days after subculturing may also be used; however, viability will decrease significantly, particularly if the cells have reached high confluence.
2. Preparing Consumables
Label the cryovials in advance based on the estimated number of vials required (include information such as cell name, passage number, date of cryopreservation, and operator).
Prepare the appropriate number of controlled-rate freezing containers (e.g., Mr. Frosty) and verify that the isopropanol level meets requirements (isopropanol should be replaced regularly—typically every 5 uses or every 3 months).

Cryopreservation Procedure
1: Cell Dissociation and Collection
Dissociate cells using EDTA according to standard subculture protocols; collect the cell suspension and centrifuge (150 × g, 3 minutes). Discard the supernatant and resuspend the cell pellet in fresh E8 medium. Set the resuspension volume to half of the final total cryopreservation volume (leaving space to add an equal volume of DMSO working solution).
2: Preparing 20% ​​DMSO Working Solution
Prepare the 20% DMSO working solution by mixing 8 parts E8 medium with 2 parts DMSO; prepare fresh before use. DMSO is liquid at room temperature and does not require heating; mix gently during preparation to avoid vigorous agitation that could generate air bubbles.
3: Adding Cryoprotectant
Slowly add an equal volume of 20% DMSO working solution dropwise to the cell suspension while mixing gently, bringing the final DMSO concentration to 10%. Do not add the solution too rapidly, as this may cause localized DMSO..." Excessive concentration causes osmotic damage to the cells.
4: Aliquoting into cryovials
Gently mix the cell suspension, then rapidly aliquot it into pre-labeled cryovials and tighten the caps. The recommended cell count per vial is 1 × 10⁶ - 5 × 10⁶ cells (or adjust according to experimental requirements).
5: Controlled-rate freezing
Place the cryovials into a controlled-rate freezing container (e.g., Mr. Frosty), ensuring each vial is surrounded by isopropanol. Place the container in a −80°C ultra-low temperature freezer; the isopropanol facilitates a controlled cooling rate of approximately 1°C/min. Freeze overnight (for at least 4 hours).
6: Transfer to liquid nitrogen for long-term storage
The following day, remove the cryovials from the −80°C freezer and rapidly transfer them to a liquid nitrogen tank (vapor or liquid phase) for long-term storage.
Note: hPSCs can be stored at −80°C for a short period (several months), but cell viability gradually declines over time; therefore, −80°C storage is not recommended for long-term preservation. The transfer process must be rapid to prevent partial thawing caused by prolonged exposure to room temperature.

Verification of cryopreservation quality
After freezing, it is recommended to randomly select one vial for a recovery test; follow the standard thawing protocol and calculate cell viability. If viability is suboptimal (e.g., below 40%), investigate potential issues in the cryopreservation process (such as cell condition, DMSO concentration, or cooling rate) and re-freeze the cells if necessary.

Reference for cell yield
Typically, one well of a 6-well plate at 70%-80% confluence yields enough cells for 2-3 cryovials. Actual yield varies depending on the cell line, growth status, and dissociation efficiency.