catalogue
I. Cells
Second, conventional culture-keeping embryonic stem cells in an undifferentiated state.
culture medium
Cell recovery
Frozen cell
Gelatin coating
Cell passage
Third, differentiation in vitro.
culture medium
Culture plate coated with polyornithine/fibronectin (with or without cover glass)
In vitro differentiation method
Fourthly, preparation of transplanted cells.
cell
Pluripotent embryonic stem cells are produced in mouse blastocysts.
1. B5-ES cells (EGFP) expressing green fluorescent protein. Dr. Nagy's lab prepared it.
2.D3-ATCC； CRL- 1934。 By the time we got it, it had been passed down for about 17 generations.
3.j 1- courtesy of Dr. Jaenish's laboratory. When we got it, it was probably handed down for 7-9 generations.
4.J 1 RTTA-RTTA expression J 1 cell, provided by Dr. Jaenish's laboratory friendship.
5. YC5-ES cells expressing yellow fluorescent protein were provided by Dr. Nagy's laboratory.
Conventional culture-maintaining ES cells in undifferentiated state.
ES cell culture uses a high-sugar medium containing ESGRO (leukemia inhibitory factor) to prevent cell differentiation. Provide cells with a flat plate coated with 0. 1% gelatin as a substrate for cell adhesion. It is suggested that cells should be transferred from 80%-90% fused plate once every 2-3 days, and the ratio is 1: 8. After cell passage, differentiated cells and undifferentiated cells should be separated by inoculating cells into uncoated tissue culture plates for 2 hours before inoculating cells into 0. 1% gelatin-coated Petri dishes. Cells were cultured at 37℃, 5% CO2 and 65438 000% humidity.
culture medium
ES:
Prepare a 20-fold solution without DMEM, HS and ESGRO (this solution can also be used in EB medium-see below). Packed in 50 ml FALCON test tubes (diluted twice, 42 ml per tube) and stored at -20℃. A culture medium was prepared by adding 2 1 ml of this solution, HS and ESGRO to 450 ml of DMEM, and filtered with a 0.2μm filter membrane. Store at 4℃. Note: A bottle of DMEM is 500ml.
Store liquid
DMEM (high sugar)
Horse serum
L- glutamine (200 mm)
Memneaa (10mm)
Hpss (1 m)
β-mercaptoethanol (55 mm)
pest
Esgro
Resuscitation cell
The cells were frozen in 10% dimethyl sulfoxide (DMSO) to prevent crystal formation that would damage the cells. However, DMSO is toxic to cells, so it is very important to carry out cell resuscitation quickly.
Steps:
1. Take out a tube of cells from liquid nitrogen;
2. Put the freezing tube into a water bath at 37℃ for 2 minutes (or the solution in the tube just completely dissolves);
3. Transfer the cells to15ml Falcon test tube;
4. Add 5ml ES medium (rinse the frozen storage tube with medium);
5. Centrifuge for 3 minutes;
6. Discard the supernatant, and culture the resuspended cells with 2ml ES, blowing 10 at least;
7. Inoculate in a 6-hole or 6cm tissue culture dish coated with gelatin (see below);
8. hatch.
Frozen cell
Frozen storage solution
90% HS and 10% DMSO
Steps:
1. 1× PBS to wash the cells, leaving a little PBS in the Petri dish;
2. Collect cells with a cell scraper;
3. Transfer the cells to 15ml Falcon test tube and centrifuge for 3min;
4. Discard the supernatant and resuspend the cells in cold frozen solution (2ml for 10cm culture dish and 6-7ml for 15cm culture dish. )
5. Sub-package in freezing tubes, each tube is1ml;
6. Leave it overnight at -80℃ and move it into liquid nitrogen the next day.
Gelatin coating
Prepare 500ml 0. 1% gelatin solution.
1. Dissolve 0.5g gelatin in 500ml PBS without calcium and magnesium (50-65℃ water bath15-30min).
2. The solution should be filtered with a 0.2μm filter membrane without cooling and stored at 4℃.
Coated Petri dish or Petri dish
1. Add enough gelatin solution to cover the culture plane (2ml of 15cm culture dish, 0.5 ~ 1ml of 10cm culture dish). The amount of solution is not important, as long as it can completely cover the culture surface. );
2. Leave it at room temperature for 30 minutes;
3. Take out the gelatin solution and store the culture plate in a packaging bag at room temperature. It is best to make the flat plate square to prevent gelatin from polluting the lid and flowing out of the culture plate.
Cell passage
It is recommended that cells be passaged every 2-3 days. Excessive growth of cells will reduce the natural differentiation rate of cells. We established a purification method to remove differentiated cells. Before seeding cells on gelatin-coated culture plates, differentiated cells were removed by adhering them to uncoated tissue culture plates. The purification step is included in the following method.
1. Take out the culture solution;
2. 1× calcium-magnesium-free PBS washing;
3. Add 1× trypsin EDTA (10× trypsin EDTA diluted with PBS). ) Incubate at 37℃ for 5 minutes.
4. Adding ES medium to inactivate pancreatin;
5. Transfer the cells to a 15ml centrifuge tube and centrifuge for 3min; ;
6. Remove the supernatant, resuspend the cells in 2 ml culture medium, and blow for at least 10-20 times.
If the amount of medium added is small, it will be easier to separate cell clusters. ES cells are easy to aggregate, so it is very important to separate them carefully during passage. This may reduce the natural differentiation of cells.
7. Inoculate cells in an uncoated tissue culture dish (using the same specifications as at the beginning) and place them in an incubator for 2 hours (during which differentiated cells will adhere while ES cells will remain suspended);
8. Transfer the culture medium containing cells to a tissue culture dish coated with gelatin (see below). Blow air to ensure that the cells are dispersed (as mentioned earlier, embryonic stem cells tend to aggregate into clusters)
9. It is suggested to adopt 1: 4- 1: 10 proportional traffic (ATCC).
It is very important to keep the passing rate of cells. In our experience, the ratio of 1: 8 is good, which can minimize the natural differentiation of cells. When using culture plates with different specifications for cell passage, table 1 can be used to calculate the passage ratio.
Differentiation in vitro
Pluripotent embryonic stem cells can differentiate into various types of cells in vitro and in vivo. Our in vitro differentiation method is beneficial to select neural precursor cells in minimal medium (step 3), expand in the presence of bFGF (step 4) and finally differentiate in step 5. Cells were cultured at 37℃, 5% CO2 and 65438 000% humidity.
Timetable (total time is 27 ~ 34 days)
The second step; 4+ 1 day step 3; 4- 10 day step 4; Step 5 within 4 days; 10- 15 days
culture medium
EB
Prepare a 20-fold solution without DMEM, FBS and ESGRO (this solution can also be used in ES medium-see above). Packed in 50 ml FALCON test tubes (diluted twice, 42 ml per tube) and stored at -20℃. 2 1 ml of this solution and 50 ml of fetal bovine serum were added to 450 ml of DMEM to make a culture medium, which was filtered with a 0.2μm filter membrane. Store at 4℃. Note: A bottle of DMEM is 500ml.
Store liquid
DMEM (high sugar)
fetal calf serum
L- glutamine (200 mm)
Memneaa (10mm)
Hpss (1 m)
β-mercaptoethanol (55 mm)
Pests (p 104 u/ ml 104 μ g/ml
ITSFn and N3:
Prepare 20 times solution without DMEM/F 12. Packed in 15ml FALCON tubes (diluted to 1×, ITSFn 7.05ml, N3 12.55ml), filtered with 0.2μm filter membrane and stored at -20℃. This solution was added to DMEM/F 12 to prepare a culture medium and stored at 4℃.
Store liquid
DMEM (high sugar)
Transferrin 50 mg/ml
Insulin 5 mg/ml
Sodium selenite 300μM
Progesterone (20μM)
Putrescine (100μM)
Pests (p104u/ml s104μ g/ml)
Laminin 100 μ g/ml
Fibronectin 250 μ g/ml
Basic rhFGF, 10 μ g/ml.
* In step 4, bFGF was added to N3 medium to make the final concentration 10ng/ml.
Preparation of ITSFn and N3 culture medium preservation solution
Use sterile solvent and diluent, and filter the solution before subpackaging. If the solution is not filtered before packaging for some reason, it needs to be marked on the test tube to let others know that the solution can not be directly used for cell culture.
Storage solution solvent, storage solution, diluent and storage
Transferrin 50 mg/ml
Insulin 5 mg/ml
Sodium selenite 300μM
Progesterone (20μM)
Putrescine (100μM)
Pests (p104u/ml s104μ g/ml)
Laminin (100 μ g/ml)
Fibronectin (250 μ g/ml)
Basic recombinant human growth factor (10 μ g/ml)
Culture plate coated with polyornithine/fibronectin (with or without cover glass)
Preparation of coating solution
Poly (L- ornithine), 15 μ g/ml
75 ml of poly L- ornithine was mixed with 425 ml 1×PBS. Store at 4℃.
Fibronectin, 1 μ g/ml
0.5 ml of fibronectin was mixed with 500 ml of 500 ml 1×PBS.
Coating process:
1. Add poly -L- ornithine for at least 2-3 hours (overnight is also acceptable).
2. Aspiration of poly-L- ornithine
3. Add fibronectin for about 1-2 hours.
4. Suck out fibronectin and dry it for 30 minutes.
5. Store at 4℃
24-well plate is 200μl, and 6-well plate is 500μl l.. Shake the culture plate to ensure that the solution can cover the cover glass or the culture well. Sometimes it is necessary to shake the culture plate violently.
In vitro differentiation method
Step 1: ES media
Cell culture (ESGRO) was maintained in the presence of LIF.
Step 2: EB culture medium
It takes 4 days to form embryoids after LIF is removed by using a bacterial Petri dish.
1. Disperse and purify cells (see cell passage section above)
2. After 2 hours of purification, transfer the cells to a 50 ml Falcon tube containing ES medium, count the cells, put an appropriate volume into a 15 ml tube, and centrifuge for 3 minutes.
3. Culture the heavily suspended cells with 2mlEB, and blow 10 times at least to make single cell suspension.
4. A 15cm bacterial culture dish can inoculate 4 ~ 5× 106 cells (1 fully fused tissue culture dish is usually enough to inoculate 4 bacterial culture dishes with the same specifications).
After 5.2 days, the medium was changed.
Transfer the embryoid into a conical tube and leave it for 3-5 minutes to precipitate the cells. Discard the supernatant, resuspend the cells in fresh medium, and inoculate them into a new bacterial Petri dish.
6. On the 4th day of EB culture, the cells were transferred to an uncoated tissue culture dish (this is a cell transplantation step). In 1 tissue culture dish, place 1 bacterial culture dish, and attach embryoids to the same specification culture dish the day before the third step.
It takes 4 days to form embryoids. After cultured in EB medium for 1 day, embryoids attached to the surface of tissue culture dishes. This day is considered as the dividing line between the second step and the third step.
Step 3-ITSFn medium
Neural precursor cells were selected in basic medium.
1. After embryoids were inoculated in tissue culture dishes for one day, the medium was changed to ITSFn medium.
2. During this period, not all embryoids have adhered, so care should be taken when removing the culture medium to keep most embryoids in the culture dish.
3. Keep the cells cultured in ITSFn for about 10 days, and change the medium as needed-about every other day.
Observing the cell morphology, nerve-like cells appeared on the 4th to 7th day. When neuron-like cells can be identified, proceed to step 4. The conversion of steps should be a few days after the first clear sign of neural precursor cells, usually from day 6 to day 10 of step 3.
Step 4 -N3 culture medium++basic fibroblast growth factor
Neural precursor cells were cultured in the medium containing 10ng/ml bFGF for amplification.
Wash with 1 PBS, and add 2ml 1× trypsin (15cm 1 the amount of trypsin required for the culture dish is subject to the above-mentioned volume) (10× trypsin EDTA is diluted with PBS).
Incubate at 2.37℃ for 5 minutes.
3. Stop pancreatin activity with 4mlEB medium, transfer the cells into a conical tube for 3 ~ 5 minutes to remove the cell mass, and transfer the supernatant to a new centrifuge tube for centrifugation.
4. Suspension of cells in N3 medium containing bFGF.
5. Inoculate the cells on the cover glass coated with poly -L- ornithine/fibronectin (it is best to put the cover glass on a 24-well culture plate or a 6-well culture plate, and put 5 cover glasses in each well of the 6-well culture plate. If it is plastic, put 4 cover glasses to maximize the number of samples). Inoculate 3.5× 105/ well in 24-well plate or 1.7× 106/ well in 6-well plate.
After 6.2 days, the medium was changed.
Step 5 -N3 culture medium
Removal of bFGF induces differentiation of neural precursor cells.
1. After the cells were inoculated on the cover glass for 4 days, the medium was changed to N3 medium without bFGF.
2. Replace the liquid as needed (about every other day)
3. Immobilize cells after 5 days of differentiation10 ~/kloc-0.
Removing culture medium
PBS washing
Add 4% formalin
Leave it at room temperature for 30 minutes.
PBS washing twice
Stored in PBS. Use PBS containing 0.0 1% sodium azide for long-term storage.
Preparation of transplanted cells
Cells were transplanted 4 days after embryoid formation (corresponding to the second step of in vitro differentiation process, cells were transferred to tissue culture plates). As mentioned above in vitro differentiation, embryoids began to form 4 days before transplantation. It usually takes another day to transplant embryoids into 10cm Petri dishes.
transplant
1. Transfer the embryoid to 15ml conical tube and let it stand for 3 ~ 5min.
It is best to centrifuge the cells for 3 minutes. Letting it settle will remove more single cells (including dead cells), and these cells can be centrifuged.
2. Remove the supernatant and suspend the embryonic weight in 1×PBS without calcium and magnesium ions.
3. Centrifuge for 3 minutes
4. Remove the supernatant and add 1× trypsin (10cm Petri dish, 1ml).
5.37℃ water bath for 5 minutes.
6. Add 5mlEB of EB medium, and carefully blow about 10 times.
It is very important to handle the cells carefully, and don't blow hard when spreading.
7. Centrifuge for 2 minutes
8. Suck out the supernatant and resuspend the cells in 500μl EB medium.
9. Blow carefully with a smooth Pasteur straw for 5 times.
10. Centrifuge twice.
1 1. Suck out the supernatant and resuspend the cells in 100μl EB medium.
Transplantation of embryonic stem cells labeled with pentoxifylline nicotinate
1. Prepare 10μg/ml nicotinic acid basic dye solution (diphenyl imide in freezer 1 18).
2. Add 1mg (the minimum amount you can weigh) to 5ml EB medium (made into 200μg/ml).
3. Dilute the solution to10μ g/ml (100μ l 200μ g/ml solution and 1.9ml EB medium).
4. As described above, the cells were resuspended in 2ml of nicotinic acid-base staining solution instead of EB medium to prepare ES cell suspension.
5. Leave the suspension at room temperature (or on ice) for 30 minutes.
6. Centrifuge for 2 minutes
7. Suck out the supernatant and resuspend the cells in 2ml EB medium.
8. repeat it
9. Centrifuge for 2 minutes
10. Suck out the supernatant, resuspend the cells in 100μl EB medium and put them on ice. This is an article I recently translated about embryonic stem cells. The translation is not good, but the meaning should be accurate. Sorry, there are two forms that have not been sent. Which teacher can tell me how to edit them? Thank you very much Thank you, Ding! I also have a good post! I looked for it for a long time and finally found it. Thank you very much Thank you, it's useless. Let's get to know it first. Thanks again! Thank you. Thank you very much. Do you have the original?
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Can you paste the original English text? Or send it to my mailbox, :adjfchen@hotmail.com.
Thank you. Not bad, not bad. I am here to send some knowledge about stem cells.
The concept of stem cells
Stem cells are a kind of cells with self-renewal and differentiation potential. It includes embryonic stem cells and adult stem cells. The development of stem cells is influenced by many internal mechanisms and microenvironment factors. At present, human embryonic stem cells have been successfully cultured in vitro. Recent studies have found that adult stem cells can differentiate laterally into other types of cells and tissues, which provides a basis for the wide application of stem cells.
During embryonic development, a single fertilized egg can divide and develop into multicellular tissues or organs. In adult animals, normal physiological metabolism or pathological damage can also cause the repair and regeneration of tissues or organs. Embryo differentiation and adult tissue regeneration are the results of further differentiation of stem cells. Embryonic stem cells are totipotent and have the ability to differentiate into almost all tissues and organs. Stem cells in adult tissues or organs are generally considered to be tissue-specific and can only differentiate into specific cells or tissues.
However, this view has been challenged at present.
The latest research shows that tissue-specific stem cells also have the potential to differentiate into other cells or tissues, which opens up a broader space for the application of stem cells.
Stem cells have the ability of self-renewal and can produce highly differentiated functional cells. Stem cells are divided into embryonic stem cells and adult stem cells according to their survival stages.
1. 1 embryonic stem cells
embryonic stem cell
When a fertilized egg divides into blastocysts, the cells in the inner cell mass are embryonic stem cells. Embryonic stem cells are omnipotent, self-renewing, and can differentiate into all tissues in the body. As early as 1970, Martin Evans had isolated embryonic stem cells from mice and cultured them in vitro. Until recently, the in vitro culture of human embryonic stem cells was successful.
In addition, embryonic stem cells (ES cells) are highly undifferentiated cells. It has developmental totipotency and can differentiate into all tissues and organs of adult animals, including germ cells. The research and utilization of embryonic stem cells is one of the core issues in bioengineering. The study of ES cells can be traced back to 1950s, and the biological study of ES cells began with the discovery of teratoma stem cells (EC cells).
At present, many researches are carried out on mouse ES cells. For example, medical teams in Germany and the United States successfully transplanted glial cells cultured from ES cells into experimental mice last year. Since then, Missouri researchers have restored some limbs of paralyzed cats through mouse embryonic cell transplantation. With the in-depth study of ES cells, life scientists' understanding of human ES cells has entered a new stage. At the end of 1998, two research groups successfully cultured human embryonic stem cells, maintaining the totipotency of embryonic stem cells to differentiate into various somatic cells. This makes it possible for scientists to use human embryonic stem cells to treat various diseases. However, the research on human embryonic stem cells has caused great controversy around the world. For the sake of social ethics, some countries even explicitly prohibit the research on human embryonic stem cells. No matter from the point of view of basic research or clinical application, the benefits brought by human ES cells far outweigh the possible negative impact on ethics, so the voice of human ES cells research is also like a tide.
1.2 adult stem cells
Many tissues and organs of adult animals, such as epidermis and hematopoietic system, have the ability to repair and regenerate. Adult stem cells play a key role in it. Under certain conditions, adult stem cells either produce new stem cells or differentiate into new functional cells according to certain procedures, thus maintaining the dynamic balance between the growth and decline of tissues and organs. It was previously thought that adult stem cells mainly included epithelial stem cells and hematopoietic stem cells. Recent research shows that neural tissue that is considered non-renewable still contains neural stem cells, which shows that adult stem cells are everywhere. The problem is how to find and isolate various tissue-specific stem cells. Adult stem cells are usually located in a specific microenvironment. Mesenchymal cells in microenvironment can produce a series of growth factors or ligands, which interact with stem cells and control the renewal and differentiation of stem cells.
1.3 hematopoietic stem cells are fine.
Hematopoietic stem cells are the only source of various blood cells in the body, which mainly exist in bone marrow, peripheral blood and umbilical cord blood. At the beginning of this year, Pang Wenxin of Institute of Hematology of Peking Union Medical College found stem cells with hematopoietic potential in muscle tissue. Hematopoietic stem cell transplantation is the most effective method to treat hematological diseases, congenital hereditary diseases and multiple and metastatic malignant tumor diseases.