PSC CDPro Medium with Supplement

PSC CDPro Medium with Supplement is a serum-free, chemically clear, and no feeder layer cells required (can be used in combination with AuctuCel matrix gel). A culture medium specially formulated for the growth and expansion of human pluripotent stem cells (PSC), which can maintain the growth activity and pluripotency of various iPSC and ESC cell lines.

1. Strict sterile environment: It is essential to ensure that the laboratory, laminar flow hood and incubator are clean and sterile. Regular cleaning and daily sweeping should be carried out.

2. Standardized operation method: Please operate strictly in accordance with the instructions.

3. Human pluripotent stem cells have limited proliferation ability in vitro and cannot maintain their differentiation potential for a long time. We suggest using cells of generations P10 to P30 for scientific research as much as possible.

4. When culturing cells, it is necessary to strictly control the cell density. PSC cells growing too densely are prone to differentiation or cause cell senescence. It is recommended that they be subcultured or frozen with a fusion degree of about 80%.

1. Culture medium preparation:

Preparation of PSC CDPro Medium: Thaw the PSC CDPro Medium Supplement at room temperature, mix well by pipetting, and then add the Supplement to the PSC CDPro Medium to prepare a complete medium. PSC CDPro Medium can be stably stored at 2-8 ℃ for 4 weeks.

Note: The PSC CDPro Medium Supplement needs to be thawed at room temperature. Do not thaw it at 37 ℃.

Before use, please preheat the complete medium required for the day at room temperature. Do not preheat the medium at 37 ℃.

2. Coated cell culture dishes/bottles:

1) Thawing of AuctuCel Matrigel:

Transfer AuctuCel Matrigel from -20 ℃ to 4 ℃ for overnight thawing (It is recommended to place AuctuCel Matrigel on ice and then thaw it overnight in a 4 ℃ refrigerator together). After the Matrigel melts, shake it with a vortex to ensure uniform dispersion of the Matrigel. Aliquot Matrigel according to the required dosage and store it in a -20 ℃ refrigerator. Avoid repeated freezing and thawing (Matrigel will gradually solidify above 10 ℃ after melting. Keep Matrigel on ice throughout the experiment).

2) The usage method of AuctuCel Matrigel:

1) Prepare the working stock solution: Dilute Matrigel at a usage concentration of 1:50 using sterile pre-cooled DMEM/F-12.

Note: It may be necessary to determine the optimal dilution of Matrigel solution for each cell line. Conduct a pre-experiment with various dilutions ranging from 1:15 to 1:50 to select the optimal concentration.

2) Coat the entire surface of the petri dish with the diluted Matrigel solution.

3) Incubate the coated petri dishes in an incubator at 37 °C and 5% CO2 for 1 hour.

Note: If the Petri dish is not used immediately, it can be stored at 2-8 ℃ and used within one week.

4) Take the coated petri dishes out of the incubator, place them in the laminar flow hood to aspirate the coating solution, draw out the diluted Matrigel solution and discard it. After removal, there is no need to rinse the petri dishes, and they can be used immediately.

5) Precautions:

The full melting of Matrigel requires at least 4 ℃ overnight. In some cases, if the product concentration is very high, it will take a longer time to melt the glue.

During the entire operation process, all culture vessels, consumables and culture media that come into contact with Matrigel should be pre-cooled, as Matrigel will start to gel when the temperature exceeds 10 ℃.

The usage concentration of Matrigel should not be lower than 3 mg/mL, and the optimal concentration for gel formation is 6-8 mg/mL.

For your safety and health, please wear a lab coat and disposable gloves when operating.

3. Cell resuscitation and culture (taking T25 Flask as an example):

1) Preheat the complete medium at room temperature.

2) Add more than 5 mL of complete medium to a 15 mL centrifuge tube, then add ROCK inhibitor to the complete medium. Generally, 10 μM Y27632 is selected, mix well and set aside for future use.

3) Take the cells out of liquid nitrogen and immediately place them in a 37 ℃ water bath, shaking rapidly to make the cryopreservation solution melt quickly.

Note:

1) During the melting process, shake the cryotubes rapidly to ensure that the cryoliquid melts quickly and evenly.

2) When shaking, be sure to avoid water covering the pipe cover to prevent contamination.

4) Wipe the outer surface of the cryotube with 75% medical alcohol.

5) Open the cryopreservation tube in the lampline bench, use a pipette to draw the cell cryopreservation suspension, transfer it to a new 15 mL centrifuge tube, and then add 2 mL of the prepared complete medium containing ROCK inhibitor.

6) Wash the cryopreservation tube once with 1 mL of complete medium containing ROCK inhibitor to collect the residual cells and reduce cell loss.

7) Centrifuge the cell suspension at 300×g for 5 minutes.

8) Centrifuge and then remove the supernatant. Add 1 mL of complete medium containing ROCK inhibitor, gently pipette the cell precipitate, fully disperse and mix it evenly, and then perform cell counting.

9) Inoculate cells in accordance with a certain number of cells into one Matrigel-coated T25 culture flask or culture container with an equivalent base area (for details, see the AuctuCel Matrigel coating steps). Add an adequate amount of complete medium containing ROCK inhibitors (the total amount of medium in one T25 culture flask should be no less than 5 mL) for culture.

10) Shake the cells well and place them in a CO2 incubator at 37 ℃, 5%CO2 and saturated humidity.

Note: Do not move or observe the cells within 2 hours after inoculation. This will seriously affect cell adhesion, leading to poor cell condition, cell agglomeration, uneven adhesion and other situations.

11) After resuscitation for 18 to 24 hours, observe the cell status and replace the cells with fresh complete culture medium as needed.

12) Change the fresh complete medium every day until the cells grow to about 80% of the fusion degree, at which point they need to be subcultured or frozen.

4. Cell passage (taking T25 Flask as an example) :

PSC cells were passaged using 0.02% EDTA solution (Versene solution) :

1) Coat Matrigel in a laminar flow hood and place the coated petri dishes in a 37 ° C incubator for about 1 hour.

2) Preheat the required amount of PSC complete medium at room temperature until it no longer feels cold to the touch.

Note: Do not preheat the culture medium in a 37 °C water bath.

3) Remove the cells, discard the cell supernatant, and rinse the container twice with Dulbecco's PBS (DPBS) free of calcium and magnesium ions.

4) Add 2 mL of 1×0.02% EDTA solution (Versene solution) to the cell culture flask.

5) Incubate at 37 ℃ for 4 to 5 minutes or at room temperature for 5 to 8 minutes. Digestion is complete when cells start to separate and round and cavities are observed in the colony under a microscope.

Note: In larger containers or certain cell lines, this process may take more than 5 minutes.

6) Aspirate 0.02% of EDTA solution (Versene solution).

7) Add 4 mL of preheated PSC complete medium containing ROCK inhibitor to the petri dish.

8) Gently blow the cells apart and collect them in a 15 mL centrifuge tube, while being careful not to produce air bubbles.

Important note: Do not scrape cells from the culture dish.

9) Add an appropriate amount of preheated PSC complete medium containing ROCK inhibitor to the coated T25 culture bottle, and then transfer an appropriate amount of cell suspension to the culture flask containing preheated PSC complete medium containing ROCK inhibitor in the required division ratio.

10) Move the container several times in the shape of an "8" to disperse the cells on the surface of the container.

11) Gently place the petri dish into a 37 ℃ incubator with 5% CO2.

12) Change the fresh PSC complete medium every day.

5. Cell Cryopreservation:

1) When the cells grow to a density suitable for passage, they can be prepared for cryopreservation.

2) For cell digestion, please refer to 4. Cell passage steps.

3) After centrifugation, remove the supernatant and resuspend the cells with an appropriate amount of stem cell cryopreservation solution.

4) Aliquot the cells into cryotubes in proportion or quantity.

5) Conduct program cryopreservation.

6) Transfer the cells to liquid nitrogen in a timely manner for long-term preservation.

Note: Cells should not be stored for a long time in a refrigerator at -80 ℃. We suggest that the storage time in a refrigerator at -80 ℃ should not exceed 48 hours.