Human embryonic stem cell passaging culture
Human embryonic stem cell passaging culture
Embryonic stem cells are cells with a self-renewal and totipotent differentiation ability to develop and differentiate all tissues and organs of the adult body, and they are the most ideal model for in vitro studies of developmental regulatory mechanisms, as well as the most ideal seed cells for use in stem cell therapy of human diseases and organ and tissue transplantation. The establishment of new human embryonic stem cell lines remains highly ethical controversial, and current research on human embryonic stem cells is largely based on studies of established human embryonic stem cell lines.
Principle
The principle of human embryonic stem cell passaging culture is the rapid growth and expansion of human embryonic stem cells, and the cell culture incubator provides exactly the environment needed for culture.
Appliance
Embryonic stem cells are commonly used to develop and differentiate all tissues and organs of the adult body, and are the most ideal model for in vitro studies of developmental regulatory mechanisms, as well as the most ideal seed cells for stem cell therapy of human diseases and organ and tissue transplantation.
Operation method
Human embryonic stem cell passaging culture
Principle
The principle of human embryonic stem cell passaging culture is the rapid growth and expansion of human embryonic stem cells, and the cell culture incubator provides exactly the environment needed for culture.
Materials and Instruments
Equipment: 37 ℃ water bath, disposable sterile Petri dishes, disposable sterile pipettes Move The basic process of human embryonic stem cell passaging culture can be divided into the following steps: (i) Reagent preparation 1. Collagenase solution is prepared at a concentration of 1 mg/mL. 30 mg of collagenase and 30 mL of DF12 medium are used to fully dissolve collagenase in DF12 medium. Remove bacteria by filtration with a 0.22 μm filter. 2. 2. Preparation of embryonic stem cell culture medium DF12 medium, 200 mL; serum substitute, 50 mL; 200 mmol/L - glutamine + 2-mercaptoethanol solution, 1.25 mL; non-essential amino acid 100x solution, 2.5 mL; b-FGF solution, 5 mL. All components were added to 250 ml culture flasks and filtered through a 0.22 μm filter to remove bacteria. The medium should preferably be used within 2 weeks. 3. 200 mmol/L -glutamine + 2-mercaptoethanol solution 200 mmol/L -glutamine, 5 mL; 2-mercaptoethanol, 7 μl, mix well. 4. b-FGF preparation b-FGF, 10 μg; 0.1% BSA aseptic, 5 mL dissolved and frozen in 0.5 mL portions. (ii) Determine the timing of transmission Generally, embryonic stem cells need to be passaged in the following cases 1. mouse embryonic fibroblasts (MEF) feeder layer is more than 2 weeks. 2. the clonal density of embryonic stem cells is too high or the clonal size is too large. 3. differentiation of embryonic stem cell clones. (iii) Collagenase treatment 1. When it is necessary to pass on the embryonic stem cells, take out the culture plate from the incubator and aspirate the culture supernatant. 2. 2. For 6-well plate culture, add 1 mL of collagenase solution to each well. 3. 3. Process for at least 5 min. 4. 4. Observe under an inverted microscope until the cell clones detach from the plate surface. Note: Wrinkles can be seen around the clones. Depending on the digestion condition, the collagenase treatment time can be extended for another 6~10 min. 5. 5. Scrape cells ① Use a glass pipette to scrape the cells from the surface of the culture plate. ② Blow the collagenase solution gently at the same time so that the cells are completely detached from the top of the culture plate. ③ All cell clusters remain in the culture plate until the entire 6-well plate has been processed according to steps (1) and (2). Note: These steps can be done under direct vision or under a dissecting fiberoptic scope. (v) Collect and break up clones 1. Transfer the cells from the entire 6-well plate into a 15 mL centrifuge tube. 2. 2. Rinse each well with 1 mL of Embryonic Stem Cell Medium and transfer the rinse solution to the cell suspension. 3. 3. In the 15 mL centrifuge tube, gently blow the cell suspension for several minutes to further break up the cell clones. Note: Avoid air bubbles when blowing. (vi) Centrifugation 1. Centrifuge the dispersed cell clones at 200 g for 5 min. 2. 2. Remove the supernatant. 3. Gently pat the cell clusters apart, add 2~3 mL of embryonic stem cell medium to resuspend the cells, and mix gently. 4. 4. Centrifuge at 200 g for 5 min. 5. At the same time of centrifugation of embryonic stem cells, take out the pre-prepared feeder plate and aspirate the MEF medium. 6. 6. Add 1 mL of PBS to each well of the feeder layer cultured in a 6-well plate and shake gently to remove the serum contained in the medium. Note: Do not treat fibroblasts with PBS for more than 6~10 min. 7. 7. After centrifugation of the embryonic stem cells, discard the supernatant and gently pat the cell mass apart. 8. Add 2~3 mL of embryonic stem cell medium and resuspend the cells. 9. 9. Add enough embryonic stem cell medium to make the volume of cell suspension in each well of the 6-well plate 2.5 mL. 10. 10. Remove the PBS from the feeder plate and add 2.5 mL of cell suspension to each well and mix gently with a pipette. 11. 11. On a horizontal table, shake the plate briefly up and down, left and right, so that the added embryonic stem cells are evenly distributed on the feeder layer. 12. Place the cultured embryonic stem cells into the incubator to reapproximate the clones. Note: During the period of reapplication, switch the incubator on and off as little as possible to maintain the stability of the culture environment for the embryonic stem cells to reapply to the wall. 13. Replace the fresh medium every day. Observe the growth status of the clone and pass it on again when needed according to the above steps. (vii) Freezing 1. Collect the cell suspension of embryonic stem cells according to the above steps and centrifuge at 200 g for 5 min. 2. 2. Prepare the cryopreservation solution (90% fetal bovine serum, 10% DMSO) on ice and set aside. 3. 3. Discard the supernatant, loosen the cell mass and resuspend it with the cryopreservation solution, add the cryopreservation solution drop by drop and mix well. Generally, 6 cells are frozen in a 6-well culture plate, and 6 mL of freezing solution is added to the cells in each plate. 4. 4. Put 1 mL of cell suspension into 1.5 mL freezing tube quickly. Cap tightly and place in an isopropyl alcohol freezer box. 5. 5. Bring the box down to room temperature, then transfer it to -80 ℃ refrigerator and keep it overnight. 6. 6. Transfer the frozen cells to liquid nitrogen tank for storage on the next day. Note: Human embryonic stem cells should not be digested into single cells before freezing, and the digested cell mass should be slightly larger. (H) Resuscitation 1. Remove the frozen embryonic stem cells from liquid nitrogen, immerse them in a 37 ℃ water bath, and gently rotate them, taking care that the lid of the freezing tube is not submerged in water. 2. When only one small ice crystal remains, remove the tube from the water bath. 3. 3. Sterilize the tubes by immersing them in a 95% ethanol bath and allow them to dry in a sterile ultra-clean bench. 4. Transfer the cells from the freezing tube to a 15 mL centrifuge tube, add 4 mL of embryonic stem cell medium drop by drop and mix gently. 5. 5. Centrifuge at 200 g for 5 min to remove the cryopreservative. 6. Remove the supernatant. 6. Remove the supernatant, gently pat the cell mass apart, and resuspend the cells by adding 2.5 mL of embryonic stem cell medium. 7. 7. Transfer the cell suspension to a 6-well plate with irradiated feeder cells. Add 2.5 mL of cell suspension per well. The feeder cells are washed with 1 mL of PBS to remove serum. 8. 8. Place the cells in the incubator, and the next day, aspirate the dead cells floating on top of the cells and change the solution, and continue the culture in the usual way. 9. 9. Observe the cell density every day and passivate as needed. Caveat 1. All reagents should be documented with shelf life and lot number and stored in small packages to avoid repeated freezing. The culture medium should be used up within 2 weeks after preparation. 2. There are various choices of digestion solution for embryonic stem cell clones, except for collagenase digestion introduced above, 0.05% trypsin digestion can also be used, the digestion time is shorter than that of collagenase digestion, generally 1~2 min, when there are wrinkles around the clone, MEF medium should be added to abort the action of trypsin, and the other steps are basically the same. 3. When using digestion to pass on, make sure not to digest the human embryonic stem cells into single cells, otherwise the rate of clone formation is very low. 4. The cell density is very important when transmitting, the general transmitting ratio of human embryonic stem cells is 1:5~1:10, the inoculated cells are too small to grow easily, it is better to transmitting once in 4~6 d. 5. human embryonic stem cells are very sensitive to the growth of human embryonic stem cells. 5. Human embryonic stem cells have high nutritional requirements, so the liquid must be changed every day, and the cells are usually cultured in 6-well dishes. 6. Generally, the 2nd~4th generation of mouse fibroblasts (MEF) are used as feeder cells. Generally, the cells are used within 3 d after inoculation into the culture dish, and the feeder cells that have been left for too long are not conducive to the growth of human embryonic stem cells. 7. The culture environment for human embryonic stem cells is 37 ℃, saturated humidity, 5% CO2The culture environment for human embryonic stem cells is 37 ℃, saturated humidity, 5% CO The incubator should be cleaned and balanced once a month, and it is better not to culture other kinds of cells in the same incubator to avoid cross-contamination. For more product details, please visit Aladdin Scientific website.
T75 culture flask, 15 mL centrifuge tube, syringe and needle, 6-well plate coated with gelatin.
Reagents and materials: pre-prepared feeder cells, 95% ethanol, and a variety of other reagents.