CN114196621A - Culture method for inducing pluripotent stem cells to have feeder layer and application - Google Patents
Culture method for inducing pluripotent stem cells to have feeder layer and application Download PDFInfo
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Abstract
The invention provides a culture method for inducing pluripotent stem cells to have feeder layers and application thereof, wherein the culture method comprises the following steps: 1) adding gelatin into a culture bottle, shaking to cover the bottom surface, placing in a 37 deg.C incubator for at least 15min, adding MEF complete culture solution heated to 37 deg.C in water bath, making recovered MEF feeder layer cells into single cell suspension, passaging to P3 generation, and adding into the culture bottle; 2) preparing the recovered iPS cells into single cell suspension, transferring the single cell suspension into a culture plate with spread MEF feeder layer cells, placing the culture plate at 37 ℃ and 5% CO2Continuously culturing in an incubator; 3) digesting the subcultured iPS cells with 0.125% pancreatin, preparing single cell suspension with a mouse embryonic stem cell culture solution containing no MEF, inoculating into a culture dish pre-coated with 0.1% gelatin, incubating at 37 deg.C for 50min to remove MEF, transferring the supernatant into a culture plate, and culturing to obtain the final productPreparing EBS embryoid bodies.
Description
Technical Field
The invention belongs to the technical field of induced pluripotent stem cell culture, and relates to a culture method of induced pluripotent stem cells with a feeder layer and application thereof.
Background
Induced pluripotent stem cells (iPS cells) were originally one cell type similar to embryonic stem cells and embryonic APSC pluripotent cells obtained by transferring a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into differentiated somatic cells using a viral vector in 2006 and reprogramming the cells, using a japanese scientist shanzhongyamiana (Shinya Yamanaka). Subsequently, various scientists around the world successively discovered other methods to produce such cells as well.
The process of establishing iPS cells mainly comprises the following steps:
(1) isolating and culturing the host cell;
(2) introducing a plurality of pluripotency-associated genes into a host cell by virus-mediated or other means;
(3) planting the virus infected cells on feeder layer cells, culturing in a special culture system for ES cells, and adding corresponding small molecular substances to promote reprogramming according to requirements during culture;
(4) identification of iPS cells (in terms of cell morphology, epigenetics, in vitro differentiation potential, etc.) was performed after ES-like cloning occurred.
Disclosure of Invention
The invention provides a culture method for inducing pluripotent stem cells to have feeder layers and application thereof, and can be applied to a rat cerebral ischemia model and cell therapy research thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a culture method for inducing pluripotent stem cells to have a feeder layer, which comprises the following steps:
1) adding gelatin into culture flask, shaking to cover bottom surface, placing in 37 deg.C incubator for at least 15min, adding MEF complete culture solution heated to 37 deg.C in water bath, making recovered MEF feeder layer cells into single cell suspension, passaging to P3 generation, and adding into culture flask.
2) Preparing the recovered iPS cells into single cell suspension, transferring the single cell suspension into a culture plate with spread MEF feeder layer cells, placing the culture plate at 37 ℃ and 5% CO2The incubator continues to culture.
3) After the cells of the iPS cells subjected to subculture are digested by 0.125% of pancreatin, a single cell suspension is prepared by using a mouse embryonic stem cell culture solution without MEF, the single cell suspension is inoculated in a culture dish coated with 0.1% of gelatin in advance, after 50min of incubation at 37 ℃, MEF is removed, the supernatant is transferred into a culture plate for culture, and the EBS embryoid-like bodies are prepared.
Preferably, in step 1), the method for recovering MEF feeder cells and preparing a single cell suspension comprises: the MEF feeder cells were removed from the cryopreservation tubes, rapidly lysed at 37 deg.C, and the outer walls of the cryopreservation tubes were scrubbed with alcohol. And transferring the cell suspension of the frozen tube into a centrifuge tube of 2mM MEF complete culture solution, centrifuging for 5 minutes at 1000rpm, removing supernatant, adding 1 mM MEF complete culture solution, and gently blowing and beating cells for multiple times to prepare single cell suspension.
Preferably, in step 1), when MEF cells from P0 generation passed to P3 generation and MEF cells from P3 generation grew to more than 80%, the old MEF complete medium was aspirated, and fresh MEF complete medium containing mitomycin C (final concentration 10mg/ml) was added. The incubation was carried out at 37 ℃ for 3 hours in an incubator. The culture broth was aspirated and washed rapidly 4 times with PBS to remove residual mitomycin C.
More preferably, the MEF feeder cells are OSKM-1 feeder cells.
Preferably, in the step 2), the method for recovering iPS cells and preparing single cell suspension comprises the following steps: the IPSCs cell cryopreservation tube is quickly taken out of the liquid nitrogen tank, quickly dissolved at 37 ℃, and the outer wall of the cryopreservation tube is scrubbed by alcohol. And transferring the cell suspension of the frozen tube into a centrifuge tube of 5mL of iPS cell complete culture solution, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, adding fresh iPS cell complete culture solution, and gently blowing and beating the cells for multiple times to prepare single cell suspension.
Preferably, in step 3), the method for subculturing the iPS cells comprises: absorbing waste liquid, flushing with PBS once, adding 1mL of 0.25% trypsin (containing EDTA) into a culture bottle, shaking gently to cover the bottom surface with the trypsin, placing at 37 ℃ for digesting cells, digesting for 2min, adding complete culture solution of mouse iPS cells to stop digestion, centrifuging at 1000rpm for 5min, discarding supernatant, adding 1mL of complete culture solution of mouse iPS cells, blowing and beating the cells for multiple times to prepare single cell suspension, adding 5mL of complete culture solution of mouse iPS cells, blowing and beating uniformly, placing into the culture bottle with spread MEF cells,5％CO2the culture is continued in the incubator, and the times and dates of passage are indicated on the culture flask, and the liquid is changed every day.
The invention also provides application of the culture method in preparation of an experimental animal model for treating cerebral ischemia of rats.
Preferably, the application comprises: diluting the cultured and separated third generation iPS cells to 1 × 105mu.L, and injecting the mixture into a cerebral ischemia position of a rat cerebral ischemia model.
More preferably, 10. mu.l of cells are injected with a microsyringe 3.5mm to the left of bregma, 0.5mm to the front, 4.5mm deep, 3d postoperatively, in a stereotaxic orientation.
The invention has the beneficial effects that:
the cells cultured by the OSKM-1 feeder layer for the pluripotent stem cells grow better. And the influence of cell therapy on the cerebral ischemia of the rat is determined based on the construction and the detection of the cerebral ischemia model of the rat.
Drawings
FIG. 1 is a diagram (100-fold) showing the cell culture in the example of the present invention.
FIG. 2 shows rats scored 1 point and 2 points according to the present example.
FIG. 3 shows the results of TTC staining according to example of the present invention.
Figure 4 is a statistical result of infarct size in an example of the invention.
FIG. 5 shows the results of HE staining according to example of the present invention.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Examples
Induced Pluripotent Stem (IPS) cell culture with feeder layer
(I) test materials and methods
1. Experimental Material
2. Laboratory apparatus
(II) Experimental procedure
1. Revived MEF feeder layer cell and revived iPS cell
1) Adding gelatin into culture flask, shaking to cover bottom surface, placing in 37 deg.C incubator for at least 15min, removing gelatin by suction (this step can be omitted), and adding MEF complete culture solution heated to 37 deg.C in water bath. The MEF feeder cells were removed from the cryopreservation tubes, rapidly lysed at 37 deg.C, and the outer walls of the cryopreservation tubes were scrubbed with alcohol. Transferring the cell suspension of the frozen tube into a centrifuge tube of 2mM MEF complete culture solution, centrifuging at 1000rpm for 5 minutes, removing the supernatant, adding 1 mM MEF complete culture solution, gently blowing cells for multiple times to prepare single cell suspension, and adding the single cell suspension into a culture bottle.
The invention adopts an OSKM-1 feeding layer, and the specific preparation comprises the following steps:
when the MEF cells of P0 passage reached P3 passage, mitomycin C (Sigma, M0503) treatment was performed. The treated MEF cells were not proliferated any more and could be used as feeders directly.
When the MEF cells reached more than 70% of the growth of P3 passage, the old MEF complete medium was aspirated, and fresh MEF complete medium containing mitomycin C (final concentration 8mg/ml) was added. The incubation was carried out at 37 ℃ for 3 hours in an incubator.
Aspirate the broth and wash rapidly 4 times with PBS to remove residual mitomycin C.
Digest cells, count cells and freeze-save. Typically, a T25 flask is plated at 1X 106A cell.
2) The IPSCs cell cryopreservation tube is quickly taken out of the liquid nitrogen tank, quickly dissolved at 37 ℃, and the outer wall of the cryopreservation tube is scrubbed by alcohol. Transferring the cell suspension of the frozen tube to a centrifuge tube of 5mL iPS cell complete culture solution, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, and adding freshThe iPS cell complete culture solution is gently blown to beat the cells for many times to prepare single cell suspension, the single cell suspension is transferred to a culture plate with well paved MEF feeder layer cells, and the culture plate is placed at 37 ℃ and 5% CO2The incubator continues to culture.
2. Subculturing iPS cells
Absorbing waste liquid, flushing with PBS once, adding 1mL of 0.25% trypsin (containing EDTA) into the culture bottle, shaking gently to make the trypsin cover the bottom surface, placing at 37 ℃ for digesting cells, digesting for 2min, adding complete culture solution of mouse iPS cells to stop digestion, centrifuging at 1000rpm for 5min, discarding supernatant, adding 1mL of complete culture solution of mouse iPS cells, blowing and beating the cells for multiple times to prepare single cell suspension, adding 5mL of complete culture solution of mouse iPS cells, blowing and beating uniformly, placing into the culture bottle with spread MEF cells, and adding 5% CO2The culture is continued in the incubator, and the times and dates of passage are indicated on the culture flask, and the liquid is changed every day.
3. Preparation of EBS embryoid bodies
After digesting the cells with 0.125% pancreatin, a single cell suspension was prepared from the culture solution of mouse embryonic stem cells without MEF, inoculated in a petri dish pre-coated with 0.1% gelatin, incubated at 37 ℃ for 50min to remove MEF (first applied), and the supernatant was transferred to a culture plate for 24h, observed under a microscope and photographed. As shown in FIG. 1, iPS cells grown to around 10-15d began to form cell colonies.
Second, rat cerebral ischemia model construction
Experimental animal and surgical instrument
1. Experimental animals: SD rats, male, 7 weeks (180-200 g).
2. Surgical instruments:
| surgical scissors | Cutting skin and thread |
| Ophthalmic lensTweezers | 2 handle |
| Micro-scissors | 1, cutting blood vessels |
| Micro-tweezers (elbow) | 2, separating blood vessel |
| Needle holder | 1 handle |
| Drag hook | 2 are provided with |
| Vascular clamp | 1 is provided with |
| Hemostatic forceps | 2 handle |
| Needle and thread | 2-0 (skin suture) and 4-0 (ligation of blood vessels) |
| Wire bolt | (selection according to body weight) |
(II) Experimental procedure
1. The rat is fixed on an operating table, the neck is preserved and disinfected, the middle of the neck is longitudinally opened by about 3 cm (the muscle on the organ can be seen by the connective tissue under the middle of the neck), the rat is pulled open by a draw hook, a Y-shaped muscle gap area can be seen, and the carotid artery blood vessel can be seen by blunt separation.
2. The external carotid artery and the common carotid artery were ligated. The ligation position of the external jugular vein is as close as possible to the bifurcation of the internal jugular vein, so that the small branch of the external jugular vein is prevented from not being ligated, and the ligation position of the common carotid artery is about 1 cm away from the bifurcation.
3. The common carotid artery is provided with a thread for standby (used for fixing a thread plug to prevent blood leakage), and the internal carotid artery is clamped by a hemostatic clamp.
4. An oblique incision is cut in the common carotid artery near the ligature (care is taken not to cut the vessel) and the inserted wire plug is secured with the reserve wire. The hemostatic clamp is loosened to insert the wire plug into the internal carotid artery in a proper position, and the insertion depth is 2 cm. (the purchased thread bolt has a mark at the position of 2 cm, and the mark is inserted into the position of the outer part of the neck and the inner part of the neck)
5. The wound was sutured and the rat was placed on a heat-insulating pad to keep the body temperature. The wire plug is pulled out after 90-120 minutes of ischemia; the wound was sutured again.
6. Behavioral scoring after rat awakening
0 minute: no symptoms of nerve damage;
1 minute: the contralateral anterior paw cannot be fully extended;
and 2, dividing: turning to the opposite side;
and 3, dividing: pouring towards the opposite side;
and 4, dividing: the patient can not walk spontaneously and the consciousness is lost.
Rats scored 1 and 2 are shown in figure 2.
Model cell therapy experiment
Injecting the iPS cells after being cultured and separated into cerebral ischemia parts for 3 days
The method comprises the following steps: cells were diluted to 1X 105mu.L. The animals in the experimental group were injected with 10. mu.l of cells using a microsyringe 3.5mm to the left of bregma, 0.5mm to the front, and 4.5mm deep, in 3d after surgery, in a stereotaxic orientation.
(II) detection of the results
1. TTC dyeing
The method comprises the following steps:
1) before sampling the tissues (brain and heart) stained by TTC, perfusing with normal saline to remove blood in the tissues, and sampling the brain tissues to perform systemic perfusion through the heart; cardiac tissue sampling the heart was removed and repeatedly flushed with saline against the ventricles until all blood was removed.
2) Brain tissue was sampled to remove all of the brain and cerebellum (for easy sectioning). Removal of blood vessels and atrial appendage during sampling of cardiac tissue only retained the ventricles.
3) After sampling, the sample is placed on a culture dish and immediately frozen in a refrigerator at-20 ℃, but the freezing time is not too long, and the sample can be cut when the frozen tissue is just hardened (otherwise, the brain tissue is easily crushed). Placing brain tissues with the top upward; the heart is required to be placed horizontally.
4) The brain (olfactory bulb and cerebellum discarded) or heart is rapidly sliced with a surgical blade or other sharp blade into slices of about 2mm in thickness (5 or 6 slices according to experimental requirements, without special requirement to default to 5 slices).
5) After slicing, carefully transferring the tissues into a 50ml centrifuge tube, adding 2% TTC dye liquor for soaking and dyeing (the TTC dye liquor just submerges all tissues properly), placing the tissues into a 37 ℃ thermostat (or a water bath kettle) in a dark place for dyeing for 15-30min, wherein the dyeing time is not too long (the dyeing can cause too deep color), and the tissues are prevented from adhering to the wall in the dyeing process to cause uneven dyeing (the tissues are checked and gently shaken every 5min to cause more uniform dyeing).
6) After the dyeing is finished, the dyeing liquid is poured off, and the brain is fixed for 1 hour by using the fixing liquid due to the fact that the brain is soft and fragile. Heart tissue was photographed after flattening the tissue using two slides.
As a result: as shown in figure 3, TTC staining of the brain tissue of the rats in the sham operation group has no white infarct area basically, the white infarct area of the brain tissue of the rats in the model group is increased remarkably, and the proportion of the white infarct area is reduced after cell therapy. The infarction area statistics result is shown in fig. 4, compared with the sham operation group, the infarction area of the model group is obviously increased; compared with the model group, the infarct size of the cell-treated group was significantly reduced.
2. HE staining results: as shown in FIG. 5, the hippocampal cells of the rats in the sham-operated group were substantially normal, free of pathological changes, uniformly stained, and regularly arranged; the hippocampal cells of the model group are disorderly, not compact and uneven in arrangement, unclear in outline, unclear in most of the nucleoli and lost in cytoplasm; the hippocampal cells of the treatment group are closely and regularly arranged, the cells are uniformly stained, and the outline is clear.
The experimental result shows that the injection cell has a repairing effect on cerebral infarction after treatment and has a certain treatment effect on cerebral ischemia.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all the embodiments of the present invention are not exhaustive, and all the obvious variations or modifications which are introduced in the technical scheme of the present invention are within the scope of the present invention.
Claims (9)
1. A method for inducing pluripotent stem cells to have a feeder layer, comprising:
1) adding gelatin into a culture bottle, shaking to cover the bottom surface, placing in a 37 deg.C incubator for at least 15min, adding MEF complete culture solution heated to 37 deg.C in water bath, making recovered MEF feeder layer cells into single cell suspension, passaging to P3 generation, and adding into the culture bottle;
2) preparing the recovered iPS cells into single cell suspension, transferring the single cell suspension into a culture plate with spread MEF feeder layer cells, placing the culture plate at 37 ℃ and 5% CO2Continuously culturing in an incubator;
3) after the cells of the iPS cells subjected to subculture are digested by 0.125% of pancreatin, a single cell suspension is prepared by using a mouse embryonic stem cell culture solution without MEF, the single cell suspension is inoculated in a culture dish coated with 0.1% of gelatin in advance, after 50min of incubation at 37 ℃, MEF is removed, the supernatant is transferred into a culture plate for culture, and the EBS embryoid-like bodies are prepared.
2. The method for culturing feeder-induced pluripotent stem cells according to claim 1, wherein the method for reviving MEF feeder cells and preparing a single cell suspension in step 1) comprises: taking out MEF feeder layer cells from the cryopreservation tube, quickly dissolving at 37 ℃, and scrubbing the outer wall of the cryopreservation tube by using alcohol; and transferring the cell suspension of the frozen tube into a centrifuge tube of 2mM MEF complete culture solution, centrifuging for 5 minutes at 1000rpm, removing supernatant, adding 1 mM MEF complete culture solution, and gently blowing and beating cells for multiple times to prepare single cell suspension.
3. The method of claim 1, wherein in step 1), when the MEF cells from P0 passage reach P3 passage and the MEF cells from P3 passage grow to 80% or more, the old MEF complete culture solution is aspirated, fresh MEF complete culture solution containing mitomycin C is added, the culture solution is incubated in an incubator at 37 ℃ for 3 hours, the culture solution is aspirated, and the culture solution is washed with PBS 4 times to remove residual mitomycin C.
4. A method of claim 1, wherein the MEF feeder cells are OSKM-1 feeder cells.
5. The method for culturing the induced pluripotent stem cells with the feeder layer according to claim 1, wherein the method for reviving the iPS cells and preparing the single cell suspension in the step 2) comprises the following steps: taking out the IPSCs cell cryopreservation tube from a liquid nitrogen tank quickly, dissolving the IPSCs cell cryopreservation tube quickly at 37 ℃, scrubbing the outer wall of the cryopreservation tube with alcohol, transferring the cell suspension of the cryopreservation tube into a centrifugal tube of 5mL of iPS cell complete culture solution, centrifuging the cell suspension at 1000rpm for 5 minutes, discarding supernatant, adding fresh iPS cell complete culture solution, and slightly blowing and beating the cells for multiple times to prepare single cell suspension.
6. The method for culturing an induced pluripotent stem cell with a feeder layer according to claim 1, wherein the iPS cell subculturing method in step 3) comprises: absorbing waste liquid, flushing with PBS once, adding 1mL of 0.25% trypsin containing EDTA into the culture bottle, shaking gently to make the trypsin cover the bottom surface, placing at 37 ℃ for digesting cells, digesting for 2min, adding complete culture solution of mouse iPS cells to stop digestion, centrifuging at 1000rpm for 5min, discarding supernatant, adding 1mL of complete culture solution of mouse iPS cells, blowing and beating cells for multiple times to prepare single cell suspension, adding 5mL of complete culture solution of mouse iPS cells, blowing and beating uniformly, placing into the culture bottle with spread MEF cells, and placing 5% CO2The culture is continued in the incubator, and the times and dates of passage are indicated on the culture flask, and the liquid is changed every day.
7. Use of the method of claim 1-6 for culturing pluripotent stem cells with feeder layer in experimental animal model for treating cerebral ischemia in rat.
8. The application according to claim 7, wherein the application comprises: diluting the cultured and separated third generation iPS cells to 1 × 105mu.L, and injecting the mixture into a cerebral ischemia position of a rat cerebral ischemia model.
9. The use of claim 8, wherein 10 μ l of cells are injected with a microsyringe 3.5mm to the left of bregma, 0.5mm to the front, 4.5mm deep at 3d postoperatively in stereotaxic positioning.
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