实验试剂

Dulbecco’s Modified Eagle Medium (D-MEM)

Fetal Bovine Serum (FBS), ESC-Qualified

D-MEM/F-12 (1X) with GlutaMAX™-I

KnockOut™ Serum Replacement (KSR)

MEM Non-Essential Amino Acids Solution, 10 mM

β-mercaptoethanol, 1000X

Gibco® Mouse Embryonic Fibroblasts (Irradiated)

Basic Fibroblast Growth Factor (bFGF)

GlutaMAX™-I (100X)

Collagenase Type IV for enzymatic passaging or StemPro® EZPassage™ Tool for mechanical passaging

实验设备

Cell Scraper

Dulbecco’s PBS (DPBS) with Calcium and Magnesium

Dulbecco’s PBS (DPBS) without Calcium and Magnesium

Geltrex® LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix or CELLstart™ CTS™ substrate

Attachment Factor

Stem Cell Tested 0.22 μm pore size sterile filters

BD Falcon T175 TC-Flasks

37°C water bath

Appropriate tissue culture plates and supplies

实验步骤

1. Preparing MEF-Conditioned Medium (MEF-CM)

1) Cover the whole surface of each new culture vessel with Attachment Factor (AF) solution and incubate the vessels for 30 minutes at 37°C or for 1 hour at room temperature. For MEF-CM generation, a T-175 flask is recommended.

2) Using sterile technique in a laminar flow culture hood, completely remove the AF solution from the culture vessel by aspiration just prior to use. Coated vessels may be used immediately or stored at room temperature for up to 24 hours.

Note: It is not necessary to wash the culture surface before adding cells or medium.

3) Plate 9.4 × 106 Mitomycin C-treated or irradiated MEFs in a T-175 flask coated with AF and containing 30 mL of MEF medium.

4) The following day, replace the MEF medium with 90 mL of PSC Culture Medium.

5) Collect the PSC Culture Medium, now considered MEF-CM, from the flasks after 24 hours of conditioning for up to seven days in a row.

6) Each day, filter sterilize the collected MEF-CM with a 0.22 μM filter. Filtered MEF-CM can be stored at–20°C until use.

7) At the time of use, thaw the MEF-CM in a 37°C waterbath, and freshly supplement it with additional bFGF (20 ng/mL).

2. Coating Culture Vessels with Geltrex® LDEV-Free, hESC-Qualified Basement Membrane Matrix

1) Thaw a 5-mL bottle of Geltrex® LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix at 2–8°C overnight.

2) Dilute the thawed Geltrex® solution 1:1 with cold sterile D-MEM/F-12 to prepare 1-mL aliquots in tubes chilled on ice. These aliquots can be frozen at –20°C or used immediately.

Note: Aliquot volumes of 1:1 diluted Geltrex® solution may be adjusted according to your needs.

3) To create working stocks, dilute a Geltrex® aliquot 1:50 with cold D-MEM on ice, for a total dilution of 1:100.

Note: An optimal dilution of the Geltrex® solution may need to be determined for each cell line. Try various dilutions from 1:30 to 1:100.

4) Quickly cover the whole surface of each culture dish with the Geltrex® solution (refer to Table 1).

5) Incubate the dishes in a 37°C, 5% CO2 incubator for 1 hour.

Note: Dishes can now be used or stored at 2–8°C for up to a week. Do not allow dishes to dry.

6) Aspirate the diluted Geltrex® solution from the culture dish and discard. You do not need to rinse off the Geltrex® solution from the culture dish after removal. Cells can now be passaged directly into MEF-CM onto the Geltrex® matrix-coated culture dish.

Note: CELLstart™ CTS™ substrate may be substituted for Geltrex® hESC-Qualified Matrix

3. Thawing and Plating hESCs

1) Label the Geltrex® matrix-coated dish with the passage number from the vial, the date, and user initials.

2) Remove the vial of hESCs from liquid nitrogen storage using metal forceps.

Note: If the vial is exposed to ambient temperatures for more than 15 seconds between removal and thawing, transfer the vial into a container containing a small amount of liquid nitrogen.

3) Roll the vial between your gloved hands until the outside is free of frost. This should take ~10–15 seconds.

4) Immerse the vial in a 37°C water bath without submerging the cap. Swirl the vial gently.

5) When only an ice crystal remains, remove the vial from the water bath, spray the outside of the vial with 70% ethanol to sterilize, and place it in hood.

6) Pipet the thawed cells gently into a sterile 50-mL conical tube using a 5-mL sterile pipette.

7) Slowly add 10 mL of MEF-CM drop-wise to cells in the 50-mL conical tube. While adding the medium, gently move the tube back and forth to mix the hESCs. This reduces osmotic shock to the cells.

8) Rinse the vial with 1 mL of MEF-CM and add to the 50-mL conical tube with cells.

9) Transfer cell suspension into a 15-mL centrifuge tube. Centrifuge the cells at 200 × g for 5 minutes.

10) Aspirate and discard the supernatant.

11) Resuspend the cell pellet in sufficient volume of MEF-CM according to Table 2 by gently pipetting the cells up and down in the tube a few times.

12) Aspirate the excess Geltrex® solution from the prepared dish and slowly add the thawed colonies onto the dish. Move the dish in several quick, short, back-and-forth and side-to-side motions to disperse cells across the surface the dish.

13) Place dish gently into the 37°C, 5% CO2 incubator and incubate the cells overnight.

14) The next day, remove the spent medium with debris using a sterile serological pipet and transfer it into a prepared Geltrex® matrix-coated dish. You can use this dish as a backup in case there is a problem with the main dish.

15) Add fresh MEF-CM to each dish according to the volumes in Table 2. Place both plates gently into a 37°C, 5% CO2 incubator overnight.

16) Examine cells under the microscope and replace spent medium daily from both plates. If feeding more than one plate, use a different pipette for each well to reduce the risk of contamination. Colonies may not be visible for up to a week.

4. Passaging hESCs

1) Label a new Geltrex® matrix-coated dish with the cell line name, the new passage number, the date, the split ratio, and user initials. Return the dish to the incubator.

2) Under a dissecting microscope, remove differentiated colonies from the dish to be passaged.

3) Aspirate the spent medium from the dish with a Pasteur pipette, and rinse the dish once with Dulbecco’s PBS (DPBS) without Calcium and Magnesium.

4) Add Collagenase Type IV (10 mg/mL) solution to the dish containing hESCs. Adjust the volume of Collagenase Type IV for various dish sizes (e.g., 35-mm dishes require 1 mL of Collagenase IV).

5) Incubate the dish(es) for 5–7 minutes in a 37°C, 5% CO2 incubator. Note that the incubation times may vary among different batches of collagenase; therefore, examination of the colonies is needed to determine the appropriate incubation time.

Note: As an alternative to Collagenase Type IV, you may use Dispase at a concentration of 2 mg/mL and incubate the dish(es) for 2–3 minutes in a 37° C, 5% CO2 incubator.

6) Stop the incubation when the edges of the colonies are starting to pull away from the plate

7) Aspirate the Collagenase Type IV Solution with a Pasteur pipette. Remove the collagenase carefully without disturbing the attached cell layer.

8) Add MEF-CM to each dish. Use a 5-mL pipette to gently blow the cells off the surface of the dish while pipetting up and down. Make sure to pipet gently to minimize the formation of bubbles.

9) After the hESCs have been removed from the surface of the well, pool the contents of the wells into a 15-mL conical tube.

10) Using a 5-mL pipette, add MEF-CM to the dish to wash and collect any residual cells. Pipet up the medium and cells, and then add the collected cells to the 15-mL tube.

11) Pipet cells up and down gently a few times in the 15-mL tube to further break up cell colonies. Pipet carefully to reduce foaming.

Note: Avoid making a single cell suspension.

12) Centrifuge at 200 × g for 5 minutes, and then aspirate the supernatant from the hESC pellet.

13) Resuspend the pellet with an appropriate amount of MEF-CM. This is dependent on the split ratio and the number of dishes used.

14) Mix the cell suspension well with a 10-mL pipette. Be careful not to break up the colonies too much or cause bubbles in the media.

15) Add appropriate volume of cell suspension to each dish. Return the dish to the incubator.

16) Move the dish(es) in several quick, short, back-and-forth and side-to-side motions to disperse cells across the surface of the dishes.

17) Incubate cells overnight to allow colonies to attach. Replace spent medium daily.

Note: While cells are attaching, be careful when opening and closing the incubator doors to avoid disturbing the even distribution of cells on the surface of the wells.