CN111961640B - Construction method and culture system of three-dimensional differentiation model of urine-derived renal stem cells - Google Patents
Construction method and culture system of three-dimensional differentiation model of urine-derived renal stem cells Download PDFInfo
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Abstract
The invention provides a construction method and a culture system of a three-dimensional differentiation model of urine-derived renal stem cells, wherein the construction method comprises the following steps: extracting and two-dimensional cell culturing, namely extracting stem cells from urine, and then carrying out subculture on trophoblast cells by using a urine-derived renal stem cell culture solution to obtain the urine-derived renal stem cells; a three-dimensional cell differentiation step, namely adding urine-derived renal stem cells into urine-derived renal stem cell culture solution, re-suspending to obtain urine-derived renal stem cell suspension, taking a bracket material, and adding the urine-derived renal stem cell suspension for culture to obtain a three-dimensional differentiation model; the trophoblast cells are fibroblasts, and by co-culturing with the trophoblast cells, a large number of active and high-purity renal stem cells can be effectively obtained, and meanwhile, the characteristics of the renal stem cells, namely self-renewal capacity and differentiation potential, can be well maintained, and the renal stem cells can form regenerated tissues and micro-organs for transplantation treatment and drug screening in three-dimensional differentiation in a short period of time.
Description
Technical Field
The invention relates to the field of cell biology, in particular to a construction method and a culture system of a three-dimensional differentiation model of urine-derived renal stem cells.
Background
End-stage renal failure, which is caused by various kidney-related diseases or toxic substances, is a major problem that afflicts the medical community. The only effective radical treatment for renal failure is in situ kidney transplantation. While kidney transplantation is not suitable for all end-stage renal failure patients due to the limitations of donor organ shortage, complex surgery, great cost, complex complications and the like. In recent years, as an alternative treatment means for organ transplantation, the role of cell transplantation in the medical fields such as blood system diseases, autoimmune diseases, functional disorders of important organs such as heart, liver and kidney, etc. is becoming important, and the potential application value thereof has led to great progress in the field of cell transplantation research. Its advantages include wide cell source, low trauma in therapeutic course, and no immunological rejection and ethical problem caused by transplanting foreign organs.
In applications where cell transplantation approaches are used to treat acute or chronic kidney injury, the selection of seed cells is critical. Seed cells considered to have potential application value in the field of regenerative medicine at present comprise allogeneic totipotent stem cells, induced pluripotent stem cells, adult tissue specific stem cells and the like. The adult tissue specific stem cells can be obtained from autologous tissues and have the characteristic of being directionally differentiated into specific tissue organs. In addition, compared with seed cells such as allototipotent stem cells, induced pluripotent stem cells and the like, adult tissue-specific stem cells are relatively easy to obtain, have low tumorigenicity, do not have the problem of histocompatibility and ethical disputes, and have great advantages as seed cells. A kidney stem cell is a stem cell that is present in adult kidney epithelial tissue and has the function of differentiating into a mature kidney function cell. Several studies have shown that after a degree of kidney injury, a few kidney stem cells present in specific sites of the nephron can proliferate and differentiate rapidly, functioning in place of damaged mature glomerular or tubular cells, thereby achieving the goal of repairing local injury. Thus, extraction of such kidney stem cells from urine may provide ideal seed cells for cell therapy techniques. The cells can be extracted from the urine of a patient and used as autologous cells, and have the characteristics of safe and low-invasive separation process, simple preparation process, low operation cost and the like. The isolated and purified kidney stem cells have higher self-renewal capacity, can obtain enough cells after short-time culture, have stable differentiation phenotype, can meet the demands of scientific research and clinical application, and have huge application potential.
At present, a two-dimensional cell culture mode is mainly adopted for obtaining the renal stem cells from urine, the cells are in a planar structure, corresponding tissues or organs with a three-dimensional structure are difficult to form, and the repairing effect is usually realized by differentiating tissue cells forming renal tubules or glomeruli after the transplantation of the renal stem cells, so that the induction differentiation of the renal stem cells is very important. The invention utilizes a three-dimensional culture system to efficiently induce stem cells to differentiate, forms micro-organs with three-dimensional structures, and can be used for detection such as subsequent transplantation or drug screening.
Disclosure of Invention
Therefore, the invention aims to overcome the defects that kidney damaged tissues in transplantation treatment are difficult to repair due to the fact that kidney stem cells with kidney damaged structure repairing capability cannot be obtained by separating and culturing from urine in the prior art, and differentiation efficiency of the urine-derived kidney stem cells is low by inducing the urine-derived kidney stem cells in a two-dimensional culture mode, so that the invention provides a construction method and a culture system of a urine-derived kidney stem cell three-dimensional differentiation model, which can obtain urine-derived kidney stem cells and effectively induce differentiation of the urine-derived kidney stem cells into kidney epithelial cells, and form regenerated tissues and micro-organs for transplantation treatment, drug screening and the like in a short period of time.
The invention provides a construction method of a three-dimensional differentiation model of urine-derived renal stem cells, which comprises the following steps:
separating and two-dimensional cell culturing, namely extracting stem cells from urine, and then carrying out subculture on trophoblast cells by using a urine-derived renal stem cell culture solution to obtain the urine-derived renal stem cells;
a three-dimensional cell differentiation step, namely adding urine-derived kidney stem cells into urine-derived kidney stem cell culture solution, re-suspending to obtain urine-derived kidney stem cells, taking a bracket material, adding the urine-derived kidney stem cells, and culturing by using the urine-derived kidney stem cell differentiation culture solution to obtain a three-dimensional differentiation model;
the trophoblast cells are fibroblasts, the urine-derived renal stem cell culture solution comprises 200-300ml of DMEM/F12 culture medium, 20-70ml of fetal bovine serum, 0.2-2mM of L-glutamine, 1-14ng/ml of insulin, 0.1-1ng/ml of epidermal growth factor, 5-30 mu g/ml of adenine and 2-20 mu g/ml of hydrocortisone, and the urine-derived renal stem cell differentiation culture solution comprises 200-300ml of DMEM culture medium, 200-300ml of F12 culture medium, 0.2-2mM of L-glutamine, 50-300ug/ml of FGF9 and 10-70ug/ml of HGF.
Further, the trophoblast cells are derived from embryonic fibroblasts;
the embryonic fibroblasts include established fibroblasts and primary cultured fibroblasts.
Preferably, the trophoblast cells include embryonic cells that are mouse embryonic fibroblasts 3T3-J2.
Further, the scaffold material is at least one selected from collagen, sodium alginate, gelatin, agarose, matrigel, hyaluronic acid, chitosan, dextran, kidney decellularized scaffold, laminin, fibronectin and fibrin; and/or the stent material is a 3D printing stent material.
Further, the construction method comprises the following steps:
separating and two-dimensional cell culturing, namely collecting urine, centrifuging, discarding supernatant to obtain a residual liquid, adding a washing buffer solution for washing, centrifuging to obtain a cell precipitate, adding a urine-derived renal stem cell culture solution, re-suspending cells to obtain a stem cell suspension, paving a trophoblast cell at the bottom of a culture vessel, establishing a trophoblast cell culture system, paving the stem cell suspension on the trophoblast cell for culturing to obtain the urine-derived renal stem cell;
and a three-dimensional cell differentiation step, namely taking the urine-derived renal stem cells, removing trophoblast cells, digesting the urine-derived stem cells, centrifuging, discarding supernatant to obtain cell sediment, adding a renal stem cell culture solution, re-suspending to obtain urine-derived renal stem cell suspension, dripping the suspension onto a glass slide containing matrigel drops, performing three-dimensional cell culture, and replacing a culture medium with a differentiation culture medium containing 100 mug/ml FGF9 and 30 mug/ml HGF to obtain a three-dimensional differentiation model of the urine-derived renal stem cells.
Further, the construction method of the slide glass containing matrigel drop comprises the steps of taking 30-100 mu l of 100% matrigel on the slide glass to form matrigel drop, placing the matrigel drop in 37 ℃ for 30min, and then adding kidney stem cell suspension for three-dimensional culture.
Preferably, in the steps of extraction and two-dimensional cell culture, trophoblast cells are taken and added into a trophoblast cell culture medium for subculture until the number of cells is expanded to 1X 10 8 ～2×10 8 After the cells were counted, the cells were digested, centrifuged, the supernatant was discarded, and the cell pellet was collected and resuspended to give a concentration of 1X 10 6 ～1×10 7 Resuspension of each mL of cell fluid, treating with gamma ray to deactivate growth, adding 5% -30% matrigel into culture vessel, incubating, sucking matrigel and mixing according to 5×10 3 ～2×10 5 Individual/cm 2 And (3) adding trophoblast cells into the culture medium, and attaching the trophoblast cells to the culture medium to obtain a trophoblast cell culture system.
Further, in the extraction and two-dimensional cell culture steps, after the attachment of trophoblast cells, the stem cell suspension is subjected to a step of 0.1X10 5 ～5×10 5 Individual/cm 2 Is spread on trophoblast cells, cultured for 3-5 days for the first time, then subjected to cell replacement at the frequency of 1 time every 2-3 days, and subcultured to obtain urine-derived renal stem cells.
Preferably, it consists of a culture of trophoblast cells, which are growth-inactivated fibroblasts, and urine-derived renal stem cells.
Further, the trophoblast cells are derived from embryonic fibroblasts;
the embryonic fibroblasts include established fibroblasts and primary cultured fibroblasts.
Preferably, the trophoblast cells include embryonic cells that are mouse embryonic fibroblasts 3T3-J2.
The invention also provides the application of the urine-derived renal stem cell three-dimensional differentiation model prepared by any one of the construction methods or any one of the culture systems in drug screening, physiological and pathological research, cell treatment and renal tissue engineering.
The invention provides a culture method of a urine-derived renal stem cell three-dimensional differentiation model, which comprises the steps of separating and culturing two-dimensional cells, extracting stem cells from urine, and then carrying out subculture on trophoblast cells by using a urine-derived renal stem cell culture solution to obtain urine-derived renal stem cells; and a three-dimensional cell differentiation step, namely adding urine-derived renal stem cells into urine-derived renal stem cell culture solution, re-suspending to obtain urine-derived renal stem cells, taking a bracket material, adding the urine-derived renal stem cells, and culturing by using the urine-derived renal stem cell differentiation culture solution to obtain a three-dimensional differentiation model. Firstly, the invention adopts the advantages of less components, simple composition and good stability of the specific urine-derived renal stem cell culture solution, is favorable for the subsequent development of clinical-grade culture medium and products, and can gradually screen the renal stem cells by combining a trophoblast cell culture system constructed by using fibroblasts with the specific urine-derived renal stem cell culture solution in the two-dimensional cell culture process to obtain the renal stem cells with higher purity, promote the stable proliferation of the renal stem cells and well maintain the characteristics of the renal stem cells, namely self-renewal capacity and differentiation potential. In the system, the kidney stem cells can be subjected to long-term subculture for more than 10 generations in vitro, meanwhile, the characteristics of the stem cells are maintained, the stem cells grow in a cloned form, the proliferation activity is maintained, the typical kidney stem cell marking characteristics are expressed, and the kidney stem cells are also a precondition for high-efficiency induction differentiation of urine-derived kidney stem cells; and secondly, performing a three-dimensional cell differentiation step on the obtained urine-derived kidney stem cells, efficiently inducing the differentiation of the urine-derived kidney stem cells to obtain a three-dimensional differentiation model, and utilizing a three-dimensional culture system and a urine-derived kidney stem cell differentiation culture solution to efficiently induce the urine-derived kidney stem cells to differentiate so as to form regenerated tissues and micro-organs for transplantation treatment in a short period of time.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a cell morphology of urine-derived renal stem cells prepared in example 1 of the present invention.
FIG. 2 is a morphology diagram of a three-dimensional differentiation model of urine-derived renal stem cells prepared in example 1 of the present invention.
FIG. 3 is a staining chart of urine-derived renal stem cells prepared in example 1 of the present invention, wherein A is a PAX2 staining chart; b is a SOX9 staining chart; c is DAPI staining.
FIG. 4 is a staining chart of a three-dimensional differentiation model of urine-derived renal stem cells prepared in the present invention, A is an AQP1 staining chart; b is a DAPI staining chart.
FIG. 5 is a graph of kidney of cisplatin-treated and control mice after three-dimensional differentiation of transplanted kidney stem cells in vivo in accordance with the present invention; wherein A and B are cisplatin treatment groups; c and D are control groups.
FIG. 6 is a Kim1 staining chart of kidney sections of cisplatin-treated and control mice after three-dimensional differentiation of transplanted kidney stem cells in vivo in accordance with the present invention; wherein A-C is cisplatin treatment group, A is Kim1 staining chart, and B is GFP staining chart; c is a DAPI staining chart; D-F is a control group, D is a Kim1 staining pattern, E is a GFP staining pattern; f is DAPI staining.
FIG. 7 is a graph showing Caspase3 staining of kidney sections of mice from cisplatin-treated and control groups after three-dimensional differentiation of transplanted kidney stem cells in vivo in accordance with the present invention; wherein A-C is cisplatin treatment group, A is Caspase3 staining pattern, and B is GFP staining pattern; c is a DAPI staining chart; D-F is a control group, D is a Caspase3 staining pattern, E is a GFP staining pattern; f is DAPI staining.
FIG. 8 shows the cell morphology of urine-derived stem cells produced in comparative example 1 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1
The embodiment provides a construction method of a three-dimensional differentiation model of urine-derived renal stem cells, which comprises the following steps:
(1) Isolation and two-dimensional cell culture steps: taking 3 branch separation tubes, respectively adding 500 mu l of penicillin/streptomycin double-antibody solution (100X) and 50 mu l of 2.5mg/ml of amphotericin solution, taking 150ml of urine of a patient with chronic kidney disease, adding the urine into the 3 branch separation tubes on average, centrifuging at a speed of 420g for 10 minutes, discarding the supernatant, keeping about 2ml of urine at the bottom of the centrifuge tube, merging the urine in all the centrifuge tubes into 1 centrifuge tube, adding washing buffer to 50ml, blowing to resuspend bottom cell sediment, centrifuging at a speed of 380g for 15 minutes again, discarding the supernatant and adding washing buffer, repeating the process for 3 times, completely discarding the supernatant after the last centrifugation, only leaving bottom cell sediment, and adding 1ml of kidney stem cell culture medium to blow and resuspend cells to obtain cell suspension.
The washing buffer solution is prepared before use, and the formula of the washing buffer solution comprises a basal medium and an additive component, wherein the basal medium is an F12 medium, and the additive component is 6% (volume ratio) of fetal calf serum, 1% (volume ratio) of 100X L-glutamine solution and 1% (volume ratio) of 100X penicillin/streptomycin double antibody solution; the kidney stem cell culture medium comprises 225ml of DMEM culture medium, 225ml of F12 culture medium, 50ml of fetal calf serum, 1.2mM of L-glutamine, 5ng/ml of insulin, 0.5ng/ml of epidermal growth factor, 30 mug/ml of adenine and 10 mug/ml of hydrocortisone.
The 3T3-J2 cell of the mouse embryo fibroblast is taken as a trophoblast cell, and a trophoblast cell culture medium is added for subculture until the 3T3-J2 cell is expanded to 1 multiplied by 10 8 After the cells were seeded, the cultured cells were digested, centrifuged at 350g for 10 minutes, the supernatant was discarded, and the cell pellet was collected according to 1X 10 6 The cell sediment is resuspended by 30ml of trophoblast cell culture medium at the concentration of individual cells/30 ml, and gamma-ray radiation treatment is carried out after the mixture is blown and evenly mixed, and the irradiation dose is 60 Gy/time, thus obtaining the growth inactivated trophoblast cells. Then, 400. Mu.l of 20% (volume ratio) matrigel was added to a 12-well plate, incubated at 37℃for 30 minutes, and the matrigel was pipetted and removed according to a 5X 10 protocol 3 Individual cells/cm 2 The trophoblast cells were added to the bottom of the culture vessel and allowed to stand for 6 hours until they were attached. Wherein the trophoblast cell culture medium is prepared by adding 10wt% FBS and 1wt% penicillin/streptomycin double anti-solventDMEM basal medium of liquid and 1wt% l-glutamine solution.
The obtained stem cell suspension is spread on trophoblast cells, and placed at 37 ℃ and 5% CO 2 Culturing under the condition, culturing for 3 days, and then changing liquid for the first time, and changing liquid according to the frequency of 1 time every 2 days. Cell clone formation was found 14 days, indicating successful isolation, and continued to culture in a trophoblast cell culture system to obtain urine-derived renal stem cells.
The morphology of the urine-derived renal stem cells was observed by using an inverted phase contrast microscope and recorded, and the results are shown in fig. 1, wherein the urine-derived renal stem cells were clonally grown, the cloning boundary was clear, and the cells in the cloning were uniform.
(2) Three-dimensional cell differentiation: taking P3 generation urine-derived renal stem cells, cloning and growing the stem cells to 70%, digesting the cells by using pancreatin, and removing the trophoblast cells in a culture system by using a magnetic bead sorting column for removing the trophoblast cells. Centrifugation was carried out at 350g for 10 minutes, and the supernatant was discarded to collect the cell pellet. According to 1.33X10 5 The cells were resuspended in urine-derived kidney stem cell culture medium at a density of individual cells/ml to give a kidney stem cell suspension. 300. Mu.l of the kidney stem cell suspension was dropped onto a prepared 8-well slide containing matrigel drop, and cultured for 24 hours, and the medium was changed to kidney stem cell differentiation medium. Cells aggregate after 7 days of differentiation, growing as vacuolated cell spheres, showing the characteristics of typical epithelial cell differentiation. Obtaining the three-dimensional differentiation structure of the urine-derived renal stem cells.
The 8-well slide containing matrigel drops should be prepared 30 minutes before use, and the method is as follows: slide glass carrying 8 isolated cells was prepared, 50. Mu.l of 100% matrigel was rapidly dropped in the center of the cell, and left to stand at 37℃for 30 minutes until it solidified.
The morphology of urine-derived renal stem cells was observed using an inverted phase contrast microscope and recorded, and as a result, as shown in fig. 2, the cells were aggregated into spheres, with vacuoles at the center, and long fusiform edge cells, which were typical epithelial cell differentiation characteristics.
Example 2
The embodiment provides a construction method of a three-dimensional differentiation model of urine-derived renal stem cells, which comprises the following steps:
(1) Isolation and two-dimensional cell culture steps: taking a 3-branch separation tube, adding 500 mu l of penicillin/streptomycin double-antibody solution (100X) as an extraction solution, taking 150ml of urine of a patient with chronic kidney disease, adding the urine into the 3-branch separation tube on average, centrifuging at a speed of 380g for 15 minutes, discarding the supernatant, keeping about 1ml of urine at the bottom of the centrifuge tube, combining the urine in all the centrifuge tubes into 1 centrifuge tube, adding a washing buffer to 50ml, blowing and beating the cell sediment at the bottom of the resuspension, centrifuging at a speed of 420g for 10 minutes again, discarding the supernatant and adding the washing buffer, repeating the process for 2-3 times, after the last centrifugation, completely discarding the supernatant, only reserving the cell sediment at the bottom, and adding 1ml of kidney stem cell culture medium to blow and beat the resuspension cells to obtain a cell suspension. The washing buffer solution is prepared before use, and the formula of the washing buffer solution comprises a basal medium and an additive component, wherein the basal medium is an F12 medium, and the additive component is fetal calf serum 4% (volume ratio), 100X L-glutamine solution 1% (volume ratio) and 100X penicillin/streptomycin double antibody solution 1% (volume ratio); the kidney stem cell culture medium contains 225ml of DMEM/F12 culture medium, 50ml of fetal calf serum, 1.2mM of L-glutamine, 7ng/ml of insulin, 0.5ng/ml of epidermal growth factor, 10 mug/ml of adenine and 10 mug/ml of hydrocortisone.
The preparation method of the trophoblast cell culture system comprises the following steps: taking mouse embryo fibroblast 3T3-J2 cells as trophoblast cells, adding trophoblast cell culture medium for subculture, and expanding 3T3-J2 cells to 2×10 8 After the cells were seeded, the cultured cells were digested, centrifuged at 350g for 5 minutes, the supernatant was discarded, and the cell pellet was collected according to 1X 10 7 The cell sediment is resuspended by 30ml of trophoblast cell culture medium at the concentration of individual cells/30 ml, and gamma-ray radiation treatment is carried out after the mixture is blown and evenly mixed, and the irradiation dose is 60 Gy/time, thus obtaining the growth inactivated trophoblast cells. Then, 600. Mu.l of 20% (volume ratio) matrigel was added to a 12-well plate, and after incubation at 37℃for 15 minutes, matrigel was pipetted and removed according to 2X 10 5 Individual cells/cm 2 The trophoblast cells are added to the bottom of the culture vessel and cultured for at least 6 hours, and the cells are used after being attached to the wall. Wherein the trophoblast cellsThe medium was DMEM basal medium containing 10wt% fbs, 1wt% penicillin/streptomycin diabody solution and 1wt% l-glutamine solution.
The obtained stem cell suspension is spread on trophoblast cells, and placed at 37 ℃ and 5% CO 2 Culturing under the condition, culturing for 4 days, and changing liquid for the first time, and changing liquid according to the frequency of 1 time every 2 days. Cell clone formation was found in 15 days, indicating successful isolation, and continued to culture in a trophoblast cell culture system to obtain urine-derived renal stem cells.
(2) Three-dimensional cell differentiation: taking P3 generation urine-derived renal stem cells, cloning and growing the stem cells to 70%, digesting the cells by using pancreatin, and removing the trophoblast cells in a culture system by using a magnetic bead sorting column for removing the trophoblast cells. Centrifugation was carried out at 350g for 10 minutes, and the supernatant was discarded to collect the cell pellet. According to 1.33X10 5 The cells were resuspended in urine-derived kidney stem cell culture medium at a density of individual cells/ml to give a kidney stem cell suspension. Mu.l of the kidney stem cell suspension was dropped onto a prepared 8-well slide containing matrigel drop, and cultured for 24 hours, and the medium was changed to a differentiation medium. Cells aggregate after 7 days of differentiation, growing as vacuolated cell spheres, showing the characteristics of typical epithelial cell differentiation. Obtaining the three-dimensional differentiation structure of the urine-derived renal stem cells.
The 8-well slide containing matrigel drops should be prepared 30 minutes before use, and the method is as follows: slide glass carrying 8 isolated cells was prepared, 50. Mu.l of 100% matrigel was rapidly dropped in the center of the cell, and left to stand at 37℃for 30 minutes until it solidified.
Experimental example 1 identification of urine-derived renal stem cells
The experimental example provides an identification of human urine-derived renal stem cells, which comprises the following steps:
performing cell immunofluorescence staining on the P3-generation urine-derived renal stem cells prepared in the step (1) in the example 1 to identify renal stem cell markers SOX9 and PAX2. After stem cell clones grew to 20-30 cell sizes, cells were fixed with 4% paraformaldehyde for 10 min. After fixation was completed, the remaining paraformaldehyde was removed by washing 3 times for 5 minutes with PBS. Cells were permeabilized by the addition of 2.5% Triton X-100 for 5-8 min. The cells were washed 3 times for 5 minutes with PBS. Adding PBS solution containing 5% -10% donkey serum, and sealing the cells for 30-60 min. The donkey serum PBS solution was removed, PBS solution containing primary antibodies (SOX 9 and PAX 2) was added, and incubated overnight at 4 ℃. The PBS solution containing the primary antibody was removed and washed 3 times for 10 minutes each with PBS. A second antibody PBS solution containing a primary antibody capable of recognizing was added and incubated at room temperature for 2 hours. The secondary antibody PBS solution was removed, and nuclei were stained with 0.1% DAPI solution and incubated for 10 minutes. The cells were washed 3 times for 20 minutes with PBS. After sealing, the cells were observed under a microscope. As shown in FIG. 3, SOX9, PAX2 and DAPI showed positive fluorescent signals, and DAPI was a nuclear dye, indicating that the kidney stem cells in the clones each expressed the kidney stem cell markers SOX9 and PAX2 specifically.
Experimental example 2 identification of three-dimensional differentiation model of urine-derived renal stem cells
Transferring the urine-derived kidney stem cell three-dimensional differentiation model prepared in the step (2) in the example 1 into an embedding box filled with an OCT embedding agent, standing at room temperature for 30 minutes, and placing the embedding box into a refrigerator at-80 ℃ for 12 hours after the matrigel becomes transparent to solidify the embedding box. Slicing with a frozen microtome to a slice thickness of 5-10 μm. Fixing the slices in pre-chilled 100% methanol for 10 min, washing with PBS, and sealing with 10% donkey serum for 30-60 min; dripping a liquid containing the AQP1 primary antibody on slice tissues, incubating for 2 hours at room temperature, and then incubating at 4 ℃ overnight; PBS was washed, ALEX Flora-labeled secondary antibody (with DAPI dye added) was added dropwise to the slice tissue and incubated at room temperature for 2 hours. The PBS was washed 4 times for 5 minutes each. Thereafter, the mixture was dropped onto the tissue with a sealing agent, and then subjected to sealing treatment with a cover glass. Finally, observing and photographing by using a fluorescence microscope.
As a result, as shown in FIG. 4, a positive signal of AQP1 was seen in the sections, and AQP1 was a marker of tubular epithelial cells, indicating that the three-dimensional differentiation system, in which urine-derived renal stem cells induced by plating were differentiated into tubular cells.
Experimental example 3 application of three-dimensional differentiation model in urine-derived renal stem cells in drug screening
Taking the P3-generation urine-derived kidney stem cells prepared in the step (1) in the example 1, adding the GFP-expressing lentivirus, incubating overnight, and then removing the virus for culture to obtain the GFP-expressing urine-derived kidney stem cells.
8 NOD/SCID mice were taken over 8-10 weeks, and a small incision was made in the left dorsal part to expose the left kidney, and the renal artery was clamped with an arteriovenous clip. Then the kidney was cut back with a surgical blade, after wiping off blood, the medulla and renal pelvis tissues were removed, and the kidney incision was sealed with medical glue.
The GFP-expressing urine-derived kidney stem cells were resuspended in a differentiation medium, and 10. Mu.L of a Rayleigh material was added and mixed, and then transplanted into the kidney of each of the above-mentioned mice, and a three-dimensional differentiation model of the urine-derived kidney stem cells was simulated in the kidney of the mice. The kidneys were replaced after implantation and the wound was sutured. 11 days after implantation, mice were equally divided into 2 groups of 4 animals, one group was intraperitoneally injected with 20mg/kg cisplatin (cispratin) and the other group was intraperitoneally injected with the same volume of PBS as a control group. Cisplatin is a common anti-tumor drug, has strong renal toxicity, and can cause damage to renal tissues.
The kidney tissues of the mice of the cisplatin treatment group and the control group were taken 14 days after the cell transplantation, and green fluorescent signals were observed, and as a result, GFP signals were clearly observed in both the mice of the cisplatin treatment group and the control group, indicating that the transplanted urine-derived kidney stem cells were integrated in the kidneys of the mice and formed a three-dimensional differentiated structure expressing GFP, as shown in fig. 5.
After fixation with 3.7% formaldehyde, the sections were sectioned at 8 μm/piece thickness after overnight at-80℃with OCT embedding, and then the sections were subjected to fluorescent signal examination with the addition of the corresponding primary antibodies (Kim 1 and GFP) or (Caspase 3 and GFP), respectively. As shown in fig. 6 and 7, kim1 signal, which marks kidney injury, was observed in the structure formed by GFP-positive kidney stem cells in kidney sections of both cisplatin-treated and control mice, whereas caspase3 signal, which marks apoptosis, was also observed in the structure formed by GFP-positive kidney stem cells in kidney sections of cisplatin-treated mice, whereas caspase3 signal, which does not mark apoptosis, was observed in kidney sections of control mice.
Cisplatin is known to have an damaging effect on cells of the tubular ducts, and the above results indicate that when urine-derived renal stem cells differentiate into a renal epithelial structure in a three-dimensional differentiation model in vivo, signals of cisplatin can be sensed, and the signals are affected by cisplatin, thereby exhibiting an apoptotic response. Therefore, the urine-derived kidney stem cell three-dimensional differentiation model has application for drug screening.
Comparative example 1 conventional urine-derived Stem cell culture method
The embodiment provides a culture method of urine-derived stem cells, which comprises the following steps:
(1) Extraction of stem cells: taking 3 branch separation tubes, adding 500 mu l of penicillin/streptomycin double-antibody solution (100X) and 50 mu l of 2.5mg/ml of amphotericin solution as extraction solutions, taking 150ml of urine of chronic kidney disease patients, adding the urine into the 3 branch separation tubes on average, centrifuging at a speed of 420g for 10 minutes, discarding the supernatant, keeping about 2ml of urine at the bottom of the centrifuge tubes, merging the urine in all the centrifuge tubes into 1 centrifuge tube, adding washing buffer to 50ml, blowing and resuspending bottom cell sediment, centrifuging at a speed of 380g for 15 minutes again, discarding the supernatant and adding washing buffer, repeating the process for 3 times, completely discarding the supernatant after the last centrifugation, adding 1ml of kidney stem cell culture medium to blow resuspended cells, obtaining urine-derived stem cell suspension, and directly spreading the urine-derived stem cell suspension into a culture plate without trophoblast cells for culture.
The washing buffer solution is prepared before use, and the formula of the washing buffer solution comprises a basal medium and an additive component, wherein the basal medium is an F12 medium, and the additive component is fetal calf serum 6% (volume ratio), 100x L-glutamine solution 1% (volume ratio), 100x penicillin/streptomycin double-antibody solution 1% (volume ratio), amphotericin 2.5 mug/ml (mass volume ratio) and gentamicin 100 mug/ml (mass volume ratio); the kidney stem cell culture medium comprises 225ml of DMEM culture medium, 225ml of F12 culture medium, 50ml of fetal calf serum, 1.2mM of L-glutamine, 5ng/ml of insulin, 0.5ng/ml of epidermal growth factor, 30 mug/ml of adenine and 10 mug/ml of hydrocortisone.
After 11 days of culture, the morphology of the cells obtained was observed by an inverted phase contrast microscope and recorded, and as shown in FIG. 8, the morphology of the cells in the visual field was greatly different from that of the urine-derived renal stem cells obtained in example 1, and the cells were not clonally grown, were large, had poor uniformity, and were slow in proliferation rate.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (8)
1. The construction method of the three-dimensional differentiation model of the urine-derived renal stem cells is characterized by comprising the following steps of:
separating and two-dimensional cell culturing, namely extracting stem cells from urine, and then carrying out subculture on trophoblast cells by using a urine-derived renal stem cell culture solution to obtain the urine-derived renal stem cells;
a three-dimensional cell differentiation step, namely adding urine-derived renal stem cells into urine-derived renal stem cell culture solution, re-suspending to obtain urine-derived renal stem cell suspension, taking a bracket material, adding the urine-derived renal stem cell suspension, and culturing by using the urine-derived renal stem cell differentiation culture solution to obtain a three-dimensional differentiation model;
the trophoblast cells are fibroblasts, and the urine-derived kidney stem cell culture solution contains 200-300ml of DMEM culture medium, 200-300ml of F12 culture medium, 20-70ml of fetal calf serum, 0.2-2mM of L-glutamine, 1-14ng/ml of insulin, 0.1-1ng/ml of epidermal growth factor, 5-30 mug/ml of adenine and 2-20 mug/ml of hydrocortisone;
the urine-derived kidney stem cell differentiation culture solution contains 200-300ml of DMEM culture medium, 200-300ml of F12 culture medium, 0.2-2mM of L-glutamine, 50-300ug/ml FGF9 and 10-70ug/ml HGF.
2. The method of claim 1, wherein the trophoblast cells are derived from embryonic fibroblasts;
the embryonic fibroblasts include established fibroblasts and primary cultured fibroblasts.
3. The method of claim 1 or 2, wherein the trophoblast is mouse embryonic fibroblast 3T3-J2.
4. The method of claim 1 or 2, wherein the scaffold material is selected from at least one of collagen, hyaluronic acid, sodium alginate, gelatin, agarose, matrigel, hyaluronic acid, chitosan, dextran, kidney decellularized scaffold, laminin, fibronectin and fibrin; and/or the stent material is a 3D printing stent material.
5. The construction method according to claim 1 or 2, comprising the steps of:
separating and two-dimensional cell culturing, namely collecting urine, centrifuging, discarding supernatant to obtain a residual liquid, adding a washing buffer solution for washing, centrifuging to obtain a cell precipitate, adding a urine-derived renal stem cell culture solution, re-suspending cells to obtain a stem cell suspension, spreading irradiated and inactivated trophoblast cells at the bottom of a culture vessel, placing the culture vessel in a cell culture box for overnight or after a period of time, establishing a trophoblast cell culture system, spreading the stem cell suspension on the trophoblast cells for culturing to obtain urine-derived renal stem cells;
and a three-dimensional cell differentiation step, namely taking the urine-derived renal stem cells, removing trophoblast cells, digesting the urine-derived renal stem cells, centrifuging, discarding supernatant to obtain cell sediment, adding a renal stem cell culture solution, re-suspending to obtain urine-derived renal stem cell suspension, dripping the urine-derived renal stem cell suspension onto a slide containing matrigel, and performing three-dimensional cell culture to obtain a three-dimensional differentiation model of the urine-derived renal stem cells.
6. The method of claim 1 or 2, wherein, in the steps of extraction and two-dimensional cell cultureIn the step, the trophoblast cells are taken and added into a trophoblast cell culture medium for subculture until the number of the cells is amplified to 1 multiplied by 10 8 ～2×10 8 After the cells were counted, the cells were digested, centrifuged, the supernatant was discarded, and the cell pellet was collected and resuspended to give a concentration of 1X 10 6 ～1×10 7 Resuspension of each mL of the cell sap, inactivation of growth by gamma irradiation, addition of matrigel to a culture vessel, incubation, pipetting of matrigel and following 5X 10 3 ～2×10 5 Individual/cm 2 And (3) adding trophoblast cells into the culture vessel, and adhering the trophoblast cells to the culture vessel to obtain the culture vessel coated with the trophoblast cells.
7. The method of constructing a cell culture system according to claim 1 or 2, wherein, in the step of extracting and culturing two-dimensional cells, after attachment of the trophoblast cells, the stem cell suspension was prepared according to a 0.1X10 5 ～5×10 5 Individual/cm 2 Is spread on trophoblast cells, cultured for 3-5 days for the first time, then subjected to cell replacement at the frequency of 1 time every 2-3 days, and subcultured to obtain urine-derived renal stem cells.
8. The three-dimensional differentiation model of urine-derived renal stem cells prepared by the construction method of any one of claims 1 to 7 for use in drug screening or in physiopathological research.
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| CN110051694A (en) * | 2019-04-22 | 2019-07-26 | 中山大学附属第一医院 | It is a kind of urine derived stem cell preparation, its preparation and its after preparing anti-organ transplant Acute immune rejection drug application |
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