General culture - keep embryonic stem cells in undifferentiated medium, cell resuscitation, cryopreserved cells, gelatin coated cells, passage
In vitro differentiation medium coated with polyornithine/fibronectin-coated plates (with or without coverslips)
In vitro differentiation method
Note: The following R1 embryonic stem cell lines are cultured in mice, and the culture of other embryonic stem cells can be referred to. However, human embryonic stem cell culture cannot use the following protocol, and a dedicated protocol and medium are required.
General culture--maintaining ES cells in an undifferentiated state
ES cell culture uses a medium (high glucose) containing LIF (leukemia inhibitory factor) and Feed cells to prevent cell differentiation. The cells were provided with a plate coated with 0.1% gelatin as a matrix for adherent cells. It is recommended to pass the cells once every 2-3 days from a plate that reaches 80%-90% confluence at a ratio of 1:8. After cell passage, cells are inoculated by inoculation before inoculation of the cells in a 0.1% gelatin-coated dish. The differentiated cells were adhered without passing through the coated tissue culture plate for 2 hours to separate the differentiated and undifferentiated cells. The cells were cultured at 37 ° C, 5% CO 2 , 100% humidity. If it is in the feed cells, then it needs to be treated with MMC to inhibit the proliferation of the feed cells, but still retain its activity of secreting LIF factor. Feed cells are not mentioned below. The feed cells can be derived from STO cells or primary embryonic fibroblasts.
Medium
ES:
Prepare a 20 x solution containing no DMEM, HS, LIF (this solution can also be used in EB medium - see below). Dispense in a 50 ml centrifuge tube (diluted to 2 x, 42 ml per tube) and stored at -20 °C. The medium was prepared by adding 21 ml of this solution, HS and LIF to 450 ml of DMEM, and filtering through a 0.22 μm filter. Store at 4 ° C for no more than 2 weeks.
Stock solution
DMEM (high sugar)
Horse serum (HS)
L-Glutamine (200 mM)
MEM NEAA (10mM)
HEPES(1M)
å·¯-mercaptoethanol (55Mm)
PEST
LIF
Resuscitation cells are frozen in 10% dimethyl sulfoxide (DMSO) to prevent the formation of crystals, which can damage cells. However, dimethyl sulfoxide is toxic to cells, and rapid cell resuscitation is important.
step:
1. Taking a tube of cells from liquid nitrogen;
2. Place the cryotube in a 37 ° C water bath for 2 minutes (or put the solution into the tube just completely dissolved);
3. Transfer the cells to a 15ml Falcon tube;
4. Add 5 ml of ES medium (flush the frozen tube with medium);
5. Centrifuge for 3 minutes;
6. Discard the supernatant, resuspend the cells in 2 ml of ES medium, and blow at least 10 times;
7. 6-well or 6 cm tissue culture dishes inoculated in gelatin (see below);
8. Incubate.
Cryopreserved cell cryopreservation solution
90% HS and 10% DMSO
step:
Wash the cells with 1.1×PBS and leave a little PBS in the culture dish;
2. Collect cells using a cell scraper;
3. Transfer the cells into a 15 ml centrifuge tube and centrifuge for 3 minutes;
4. Discard the supernatant and resuspend the cells in a cold stock solution (2 ml for a 10 cm dish and 6-7 ml for a 15 cm dish).
5. Dispense in a cryotube, 1ml per tube;
6. At -80 ° C overnight, the next day was moved into liquid nitrogen.
Geltin (gelatin) coating is prepared with 500ml 0.1% geltin solution
1. 0.5 g of gelatin was dissolved in 500 ml of calcium-free magnesium in PBS (50-65 ° C water bath for 15 to 30 minutes).
2. It is preferred to filter through a 0.22 μm filter without cooling the solution and store at 4 °C.
Coated plate or petri dish
1. Add enough gelatin solution to cover the culture plane (15 ml culture dish plus 2 ml, 10 cm culture dish plus 0.5 ~ 1 ml, the amount of the solution is not important, as long as it can completely cover the culture surface.);
2. Set at room temperature for 30 minutes;
3. The gelatin solution is removed, and the culture plate is stored in a packaging bag at room temperature, and the plate is preferably laid flat or inverted to prevent the gelatin from contaminating the lid and flowing out of the culture plate.
Cell passage suggests that cells are passaged every 2 days, and overgrowth cells reduce the natural differentiation rate of the cells. We have established a purification method to remove differentiated cells by inoculating the cells on gelatin-coated plates. Differentiated cells adhere to uncoated tissue culture plates to remove differentiated cells. The purification step is included in the method below.
1. Removing the culture solution;
2. Calcium-free magnesium PBS (Gibco) washing;
3. Trypsin EDTA (Gibco) was added. Incubate for 5 minutes at 37 °C.
4. Incorporation of ES medium to inactivate trypsin;
5. Transfer the cells into a 15 ml centrifuge tube and centrifuge for 3 min;
6. The supernatant was removed and the cells were resuspended in 2 ml of ES medium and pipetted for at least 10-20 times.
If the amount of medium added is small, it will be easier to separate the cell mass. ES cells have a tendency to aggregate, and it is important to carefully separate the cells during the passage. This may reduce the natural differentiation of cells.
7. The cells were seeded in uncoated tissue culture dishes (using the same size of petri dish as the beginning) and placed in a thermostat for 2 hours (the differentiated cells will adhere during this time, while the ES cells will remain suspended);
8. The medium containing the cells was transferred to a geltin-coated tissue culture dish. Blowing to ensure that the cells are dispersed (as mentioned earlier - the tendency of ES cells to aggregate)
9. It is recommended to pass the ratio of 1:4-1:10 (ATCC)
It is important to maintain the passage ratio of the cells. In our experience, the ratio of 1:8 is good, which can minimize the natural differentiation of cells. When you use different sizes of plates for cell passage, you can use Table 1 to calculate the passaging ratio.
In vitro differentiation of embryonic stem cells can differentiate into various types of cells in vitro and in vivo. Our in vitro differentiation method facilitates the selection of neural precursor cells by the selection in a minimal amount of medium (step 3), in the presence of bFGF (step 4) and finally in step 5. The cells were cultured at 37 ° C, 5% CO 2 , 100% humidity. Generally, the differentiation into the direction of nerve cells is induced in vitro, and it can also be induced by using a low concentration of RA.
Timeline (total time is 27 to 34 days)
Step 2; 4+1 days, step 3; 4-10 days, step 4; 4 days, step 5; 10-15 days
Differentiation of embryonic stem cells into cardiomyocytes in vitro will naturally differentiate into cardiomyocytes in the case of removal of LIF in vitro. The R1 line of mice generally has pulsating cardiomyocytes on the 7th to 8th day, which is completely consistent with the embryonic development timeline. .
Medium
EB
Prepare a solution of 20 x without DMEM, FBS, LIF (this solution can also be used for ES medium - see above). Dispense in a 50 ml centrifuge tube (diluted to 2 x, 42 ml per tube) and stored at -20 °C. 21 ml of this solution and 50 ml of FBS were added to 450 ml of DMEM to prepare a medium, which was filtered through a 0.2 μm filter. Store at 4 °C.
Stock solution
DMEM (high sugar)
Fetal bovine serum
L-Glutamine (200 mM)
MEM NEAA (10mM)
HEPES(1M)
å·¯-mercaptoethanol (55Mm)
PEST (P104U/mlS104μg/ml
ITSFn and N3 (differentiation medium):
A 20 x solution containing no DMEM/F12 was prepared. Dispense in a 15 ml centrifuge tube (diluted to 1 ×, ITSFn 7.05 ml, N3 12.55 ml), filter through a 0.2 μm filter and store at -20 °C. The solution was added to DMEM/F12 to prepare a medium, which was stored at 4 °C.
Stock solution
DMEM (high sugar)
Transferrin 50mg/ml
Insulin 5mg/ml
Sodium selenite 300μM
Progesterone (20μM)
Putrescine (100μM)
PEST (P104U/ml S104μg/ml)
Laminin 100μg/ml
Fibronectin 250μg/ml
Alkaline rhFGF, 10μg/ml
* In step 4, bFGF was added to the N3 medium to a final concentration of 10 ng/ml.
Preparation of ITSFn and N3 medium stock solutions using sterile solvents and diluents, the solution should be filtered before dispensing. If for some reason the solution is not filtered before dispensing, it needs to be marked on the tube so that others can It is known at the time that this solution cannot be directly used for cell culture.
Stock solution Solvent, stock solution, diluent and storage transferrin 50mg/ml
Insulin 5mg/ml
Sodium selenite 300μM
Progesterone (20μM)
Putrescine (100μM)
PEST (P104U/ml S104μg/ml)
Laminin (100μg/ml)
Fibronectin (250μg/ml)
Alkaline rhFGF, (10μg/ml)
Plates coated with polyornithine/fibronectin (with or without coverslips)
Preparation of coating solution poly-L-lysine, 15μg/ml
75 ml of poly-L-lysine and 425 ml of 1 x PBS were mixed. Store at 4 °C.
Fibronectin, 1μg/ml
0.5 ml of fibronectin was mixed with 500 ml of 1 x PBS.
Coating process:
1. Add poly-L-lysine for at least 2-3 hours (overnight)
2. Poly-L-lysine
3. Add fiber binding protein for about 1-2 hours
4. Aspirate the fibronectin and let it dry for 30 minutes.
5. Store at 4 ° C
200 μl for 24-well plates and 500 μl for 6-well plates. The shaker plate ensures that the solution covers the coverslip or well. Sometimes it is necessary to violently shake the plate.
In vitro differentiation method
Step 1: ES medium maintains cell culture in the presence of LIF
Step 2: EB medium It takes 4 days to form the embryoid body after removal of LIF using a bacterial culture dish.
1. Disperse and purify cells (see cell passage section above)
2. After 2 hours of purification, the cells were transferred to a 50 ml Falcon tube containing ES medium, and the cells were counted and placed in a 15 ml tube and centrifuged for 3 minutes.
3. Resuspend the cells in 2 ml of EB medium and pipet at least 10 times to make a single cell suspension.
4. A 15 cm bacterial culture dish was inoculated with 4 to 5 x 106 cells (1 fully fused tissue culture dish is usually sufficient to inoculate 4 bacterial culture dishes of the same size).
After 5.2 days, the medium was changed and the embryoid bodies were transferred to a conical tube and allowed to stand for 3 to 5 minutes to sediment the cells. The supernatant was discarded and the cells were resuspended in fresh medium and seeded in a new bacterial culture dish.
6. On day 4 of culture with EB medium, the cells were transferred to uncoated tissue culture dishes (this is the cell transplantation step). One bacterial culture dish was placed in one tissue culture dish, and the embryoid body was adhered to the same specification plate one day before the third step.
It takes 4 days to form the embryoid body. An additional 1 angel embryoid body was adhered to the surface of the tissue culture dish in EB medium. This day is considered to be the boundary between steps 2 and 3.
Step 3 - ITSFn medium selects neural precursor cells in the least amount of medium
1. Replace the medium with ITSFn medium after the embryoid body is inoculated in the tissue culture dish for one day.
2. Not all embryoid bodies have adhered during this period, so care should be taken when removing the medium to leave most of the embryoid bodies in the culture dish.
3. The cells were maintained in ITSFn for approximately 10 days and the medium was changed as needed - approximately every other day.
The morphology of the cells was observed, and the neuron-like cells appeared approximately on the 4th to 7th day. When the neuron-like cells can be identified, proceed to step 4. The conversion of the steps should take place a few days after the first clear sign of the neural precursor cells, usually on the 6th to 10th day of the third step.
Step 4 - N3 medium + bFGF
Amplification was carried out by culturing neural precursor cells in a medium containing 10 ng/ml of bFGF.
1. Wash with PBS and add 1-2 ml trypsin
Incubate at 2.37 ° C for 5 minutes
3. The trypsin activity was stopped with 4 ml of EB medium, and the cells were transferred to a conical tube and left for 3 to 5 minutes to remove the cell mass, and the supernatant was transferred to a new centrifuge tube for centrifugation.
4. The cells were resuspended in N3 medium containing bFGF.
5. The cells were seeded on a coverslip coated with poly-L-ornithine/fibronectin (preferably placed in a 24-well plate or a 6-well plate, and placed in each well in a 6-well plate) If the coverslips are placed in plastic 4, to maximize the number of samples obtained). The 24-well plate was inoculated with 3.5 x 105/well or 6-well plate at 1.7 x 106/well.
Change medium after 6.2 days
Step 5 - N3 medium differentiates neural precursor cells by removing bFGF
1. After the cells were inoculated on coverslips for 4 days, the medium was changed to N3 medium without bFGF.
2. Change the fluid as needed (about every other day)
3. Fixed cell removal medium after 10 to 15 days of differentiation
Wash in PBS and add 4% formalin for 30 minutes at room temperature.
The PBS was washed twice and stored in PBS. Long-term storage using PBS containing 0.01% sodium azide
Prepared cells for transplanted cells were transplanted 4 days after embryoid body formation (corresponding to the end of the in vitro differentiation process, cells were transferred to tissue culture plates). Embryoids begin to form 4 days prior to transplantation as described in the in vitro differentiation. It usually takes another day to transplant the embryoid body into a 10 cm culture dish.
transplant
1. The embryoid bodies were transferred to a 15 ml conical tube and allowed to stand for 3 to 5 minutes to allow the embryoid bodies to settle.
It is better to centrifuge the cells for 3 minutes. Placement to set it off will remove more individual cells (including dead cells) and these cells can be centrifuged.
2. The supernatant was removed and the embryoid body weight was suspended in 1×PBS without calcium and magnesium ions.
3. Centrifuge for 3 minutes
4. Remove the supernatant and add 1× trypsin (10 cm culture dish plus 1 ml)
5.37 ° C water bath for 5 minutes
6. It is important to carefully pipe the cells by adding 5 ml of EB medium and carefully pipetting about 10 times. Do not blow vigorously when the cells are passaged to disperse the cells.
7. Centrifuge for 2 minutes
8. Aspirate the supernatant and resuspend the cells in 500 μl of EB medium.
9. Carefully blow 5 times with a smooth Pasteur pipette
10. Centrifugation 2 times
11. Aspirate the supernatant and resuspend the cells in 100 μl of EB medium.
Nicotine nicotinic acid labeled ES cells for transplantation
1. Prepare a 10μg/ml niacin alkaline stain (diphenylimide, in the freezer 118)
2. Add 1mg (the smallest amount you can weigh) to 5ml EB medium (made in 200μg/ml)
3. Dilute the solution to 10 μg/ml (100 μl 200 μg/ml solution and 1.9 ml EB medium)
4. The ES cell suspension was prepared by resuspending the cells in 2 ml of nicotinic acid sensitizing solution instead of EB medium as described above.
5. Place the suspension at room temperature (or ice) for 30 minutes
6. Centrifuge for 2 minutes
7. Aspirate the supernatant and resuspend the cells in 2 ml EB medium
8. repeat
9. Centrifuge for 2 minutes
10. The supernatant was aspirated and the cells were resuspended in 100 μl of EB medium and placed on ice.
Beeswax is obtained from the honeycombs of bees after the Honey has been removed by draining or centrifuging. The combs are melted with hot water, steam or solar heat; the melted product is filtered and cast into cakes of yellow beeswax. With detoxification, wound healing, tissue regeneration, pain relieving effect. Commonly used in the ulcer not healing, ecthyma erosion, ulceration of trauma, burn and scald.
Beeswax
Bee Wax, Organic Beeswax, Pure Natural Beeswax, White Refined Beeswax
Easy Food (Jiaxing) Co., Ltd. , https://www.jxeasyfood.com