WO2018228071A1 - 一种人多能干细胞来源人视网膜色素上皮细胞的制备和扩增培养方法 - Google Patents

一种人多能干细胞来源人视网膜色素上皮细胞的制备和扩增培养方法 Download PDF

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WO2018228071A1
WO2018228071A1 PCT/CN2018/085026 CN2018085026W WO2018228071A1 WO 2018228071 A1 WO2018228071 A1 WO 2018228071A1 CN 2018085026 W CN2018085026 W CN 2018085026W WO 2018228071 A1 WO2018228071 A1 WO 2018228071A1
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rpe
cells
cell
pluripotent stem
pigment
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钟秀风
葛坚
刘胜旭
彭福华
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中山大学中山眼科中心
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Definitions

  • the invention belongs to the technical field of stem cells, and particularly relates to a method for preparing, expanding and cultivating human retinal pigment epithelial cells derived from human pluripotent stem cells.
  • the retinal pigment epithelium (RPE) is located outside the retinal neuroepithelial layer, providing nutrients to the latter and participating in phototransduction reactions. Degenerative death or dysfunction is an important cause of retinal degenerative eye disease. Retinal pigment epithelial cell transplantation is one of the most promising methods of recovery, but the lack of RPE cells restricts the development of this treatment. Prior to the rise of stem cell technology, RPE cell sources were limited to isolation from early aborted embryos or eye donations donated by volunteers. Recent studies have shown that human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have a retinal pigment epithelium. The ability of cells to differentiate.
  • hPSCs human pluripotent stem cells
  • hESCs human embryonic stem cells
  • hiPSCs human induced pluripotent stem cells
  • hPSCs-induced RPE cells are the most promising seed cells for the treatment of retinal degenerative diseases.
  • the induction methods for the differentiation of pluripotent stem cells into RPE cells are roughly divided into two categories, 2D traditional adherent culture method and 3D retinal induced differentiation method.
  • the RPE obtained by these methods is usually mixed with non-RPE cells produced during hPSC differentiation. Therefore, how to separate and purify the RPE cells and obtain the RPE seed cells for related research or treatment is a technical problem that needs to be solved.
  • the object of the present invention is to overcome the deficiencies in the prior art and to provide a simple and widely applicable method for preparing, expanding and culturing human pluripotent stem cell-derived human retinal pigment epithelial cells.
  • a method for preparing human pluripotent stem cell-derived human retinal pigment epithelial cells comprises the steps of: collecting 3D-RPE spheres derived from human pluripotent stem cells, mechanically separating and removing non-RPE cells or agglomerates containing no pigment, The RPE cell sheet containing the pigment was taken, and the RPE cell sheet containing the pigment was digested to obtain a RPE single cell suspension, thereby obtaining human pluripotent stem cell-derived human retinal pigment epithelial cells.
  • the pluripotent stem cell-derived RPE cells prepared by the method of the invention have the same characteristics as the human embryo-derived RPE cells, and have typical RPE cell morphological characteristics, and express specific molecular tags PAX6, OTX2, ZO-1 and RPE65, and exhibit normal physiological functions such as Polar secretory cytokine PEDF.
  • the human pluripotent stem cell is preferably a human embryonic stem cell or a human induced pluripotent stem cell. These two cells are subjected to cell culture by a known method.
  • the 3D-RPE sphere is a human pluripotent stem cell that is induced to differentiate into retinal cells containing RPE cells, and then the adherent cells, including RPE cells, are scraped up and subjected to suspension culture to obtain 3D-RPE spheres.
  • the 3D-RPE sphere can be free or adhere to one side of the nerve retinal cup or to one side of the other cell mass.
  • the 3D-RPE sphere is preferably a 3D-RPE sphere in which human pluripotent stem cells are induced to differentiate for more than 40 days.
  • the day when hPSC initiates differentiation i.e., the expansion culture medium of hPSC is replaced with a differentiation culture solution or preparation of embryoid bodies) is set to the "0" day of differentiation.
  • the mechanical separation, removal of pigment-free non-RPE cells or agglomerates, and the removal of the pigment-containing RPE cell sheet is specifically: transferring all 3D-RPE spheres into a cell container, digesting with a digestive juice at 37 ° C water bath After 8-15 min, the digestive juice was aspirated, and after washing several times in 1 ⁇ PBS, the RPE cell sheets containing the pigment were separated from the non-pigmented non-RPE cells or pellets using a tungsten needle, and the RPE cell sheets containing the pigment were taken.
  • the digestive juice is preferably a Dispase II solution having a mass fraction of 1-2%.
  • the enzymatically digesting the RPE cell sheet containing the pigment to obtain the RPE single cell suspension is specifically carried out by transferring the RPE cell sheet containing the pigment into TrypLE Express solution, digesting in a water bath at 37 ° C for 7-10 min, and centrifuging. The digestive juice was removed, resuspended in RPE cell culture medium, and the cells were filtered through a 70-100 ⁇ m filter to obtain a RPE single cell suspension.
  • the invention also provides a method for expanding and culturing human pluripotent stem cell-derived human retinal pigment epithelial cells, which is characterized in that the above RPE single cell suspension is centrifuged, the supernatant is discarded, and then resuspended in RPE cell culture medium, and inoculated.
  • Primary culture is carried out in a cell culture vessel pre-coated with an extracellular matrix, and after the cells are overgrown, subculture is carried out to obtain human pluripotent stem cell-derived human retinal pigment epithelial cells.
  • the primary culture inoculated has a cell density of 5 ⁇ 10 4 cells/cm 2 or more .
  • the cells are subcultured and then subcultured.
  • the primary cultured cells reach 90-100%, the medium is discarded, washed with PBS, and digested with TrypLE Express in a 37 ° C incubator for 7-10 min.
  • the RPE cell culture medium was stopped for digestion, the cells were gently blown off with a pipette, the digestive juice was removed by centrifugation, the cells were resuspended in RPE cell culture medium, and the cells were seeded into a cell culture vessel pre-coated with an extracellular matrix, and subcultured.
  • the degree of cell fusion reaches 90-100%, the above steps are repeated for repeated subculture; the cell density of the subcultured inoculation is greater than or equal to 2 ⁇ 10 4 cells/cm 2 .
  • the human RPE cells obtained by the method of the present invention can be transmitted for at least 5 generations.
  • the extracellular matrix is preferably Matrigel or Gelatin.
  • the cell culture vessel is preferably a culture plate, a culture dish or a culture flask.
  • the primary culture and subculture cell culture medium are formulated as follows: 10 mL fetal bovine serum, 2 mL 50 ⁇ B27, 1 mL 100 ⁇ streptomycin mixture, 1 mL 100 ⁇ non-essential amino acid per 100 mL of cell culture medium. 1 mL of 100 ⁇ glutamine and 0.1 mL of 1000 ⁇ taurine, the balance being DMEM/F12 (3:1) mixed medium, the DMEM/F12 (3:1) mixed medium was prepared by DMEM/F12 The (1:1) medium and the DMEM medium were mixed at a volume ratio of 3:2.
  • the human RPE cells are cryopreserved, specifically, the obtained RPE primary cells and passage cells can be frozen by a known method.
  • the cryopreservation solution was a cell culture medium containing a volume fraction of 10% DMSO. Cryopreserved RPE cells can resuscitate and have the same cellular properties.
  • the pluripotent stem cell-derived human RPE cells prepared by the method of the invention have the same characteristics as the human embryo-derived RPE cells, and have typical RPE cell morphological characteristics, and express specific molecular tags PAX6, OTX2, ZO-1 and RPE65, and exhibit normal physiological functions such as The polar secretory cytokine, PEDF, therefore provides seed cell material for related research and treatment.
  • the method of the invention has the following advantages:
  • the technology is simple and convenient, mainly separating, purifying and amplifying RPE cells by mechanical separation and enzymatic digestion, and does not rely on complicated experimental equipment and techniques such as flow cytometry, magnetic bead sorting or reporter gene labeling technology.
  • the digestive solutions Dispase II and TrypLE Express used in the digestion of RPE are also mild, with little damage to RPE cells.
  • the preparation of three different hPSC-derived RPE cells has been successfully completed using the method of the invention, and each cell line was repeated at least 3 times.
  • the whole set of experimental techniques is simple and easy to learn, and can be quickly mastered by beginners, with low cost and good efficiency.
  • the hPSC-derived human RPE cells obtained by the method of the invention have good amplification and passage ability, good growth characteristics, high yield, can be obtained in batches, and reduce batch-to-batch differences.
  • the cells When seeded in a Matrigel-coated plate at a density of more than 2 ⁇ 10 4 cells/cm 2 , the cells may have a degree of fusion of more than 90% in about 7 days.
  • the RPE cells prepared by the method of the invention can be amplified and cultured, and the amplification efficiency is nearly 15 times, and the serial transmission can be more than 3000 times.
  • the RPE cell doubling days obtained by the method of the present invention are about 1.52 days, and it takes only 7 days to complete the amplification 1 generation, at least 5 passages can be passed, and the cryopreservation ability is obtained.
  • the hPSC-derived human RPE cells prepared by the method of the present invention can obtain typical RPE cell morphology and pigmentation, which is similar to the culture characteristics and cell morphology of human embryo-derived RPE cells.
  • the RPE cells prepared by the method of the present invention express the RPE-specific molecular tags PAX6, OTX2, ZO-1, RPE65, and have similar properties to human embryonic retinal-derived RPE cells.
  • RPE cells prepared by the method of the invention are similar to those of RPE cells in vivo, including cytokines such as transepithelial electrical resistance and polar secretion of PEDF, suggesting good application prospects.
  • the RPE cell prepared by the method of the invention has high purity, up to 98%, and can be widely used as a research material in related fields to solve the bottleneck problem of seed cells.
  • the present invention establishes a new technology for preparing human RPE cells from hPSC, and can obtain human RPE in batches.
  • the obtained human RPE cells are very similar to human embryonic retina-derived RPE in terms of growth characteristics, cell morphology, specific molecular expression profile and function, and have high purity, suggesting that RPE prepared by the method of the present invention is involved in tissue engineering, regenerative medicine, and disease mechanism.
  • drug screening and other related fields have broad application prospects, especially to provide seed cells for research and treatment of retinal diseases, and solve the bottleneck problem of limited sources of human retinal pigment epithelial cells and lack of RPE transplant donors.
  • the method of the invention reaches the world leading level in the same field, and has great significance for the treatment of retinal diseases patients.
  • Figure 1 is an inverted microscope picture of hPSC.
  • Figure 2 is an inverted micrograph (40x) of RPE cells formed 26 days after hPSC induced differentiation.
  • Figure 3 is an inverted micrograph (40x) of 3D-RPE spheres obtained by hPSC-induced differentiation suspension culture.
  • the 3D RPE sphere is attached to the side of the nerve retina (NR) (A) or to the side of the cell mass (B).
  • Figure 4 is a picture (100 x) of a single RPE cell obtained by mechanically separating and purifying the 3D-RPE sphere.
  • Figure 5 is a photograph (200x) of primary culture of RPE cells prepared in Example 6 for seven days.
  • Figure 6 is a photograph (100x) of the RPE cells prepared in Example 6 for the fifth subculture for seven days.
  • Figure 7 is a photograph of the results of immunofluorescence.
  • Figure 8 is a graph showing the results of transepithelial electrical resistance (TEER) detection.
  • Figure 9 is a graph showing the results of an ELISA method for examining PEDF.
  • Figure 10 shows the results of flow cytometry.
  • Example 1 Expansion and culture of human pluripotent stem cells
  • hPSC human pluripotent stem cells
  • BC1-GFP-hiPSC and BC1-hiPSC were donated to friends, and another hiPSC was purchased from Life Technology, USA ( Episomal hiPSC Line, A18945). All cells were seeded in a 6-well culture plate coated with extracellular matrix MatriGel (Corning, 354277) and expanded with mTeSR1. When the degree of cell fusion was 80%-90%, it was digested with 0.5 mM EDTA (Life, 15575-038), and subcultured with cells 1:8 to 1:12. Under the inverted microscope, the cells were flaky, colony-like growth, and the cells in the colonies were closely arranged and the boundary was unclear (Fig. 1), thereby amplifying human pluripotent stem cells (hPSC).
  • Example 2 hPSC induces differentiation into retinal cells including pigment epithelial cells
  • hPSC inducing hPSC differentiation into retinal cells including RPE cells .
  • the day when hPSC initiates differentiation i.e., the expansion culture medium of hPSC is replaced with a differentiation culture solution or preparation of embryoid bodies) is set to the "0" day of differentiation.
  • Example 3 Acquisition and culture of hPSC-derived 3D-RPE spheres
  • the retinal cells including the RPE cells at the 4th week of differentiation induced by hPSC were recognized by microscopically elevated, circular, and refractive neuroretinal (NR) and RPE.
  • RPE can grow around NR, or it can be grown individually or in sheets ( Figure 2).
  • the NR and its nearby RPE were picked up by a self-made tungsten wire needle or a 1 mL syringe, and transferred to a low-adsorption culture dish for suspension culture, and the medium was an RPE cell culture medium.
  • the medium is RPE cell culture medium.
  • the suspension cultured RPE is usually crimped into spheres (ie, 3D-RPE spheres), attached to the NR side or to the other cell mass side (Fig. 3), and may also be freed from the culture.
  • Example 4 Collecting and mechanically separating and purifying hPSC-derived 3D-RPE spheres
  • Example 5 Isolation and purification of hPSC-derived human RPE tablets
  • the purified 3D-RPE spheres of Example 4 were digested with Dispase II (Sigma, D4693-1G) at a mass fraction of 1% for 8-15 min at 37 ° C to remove the digestive juice and washed 3 times with 1 ⁇ PBS.
  • the surface of the 3D-RPE sphere is RPE, black, and the inside is a non-RPE cell mass. Under dissecting microscope, the two were dissected and separated using a tungsten wire needle, and the separated RPE cell sheets were collected.
  • the purified RPE cell pellet was treated with TrypLE Express solution (Gibco, 12604-013), digested in a water bath at 37 ° C for 7-10 min, the digestion was terminated by adding an equal volume of RPE cell culture medium, and the 1 ml pipette was used to blow the dispersed cells until no visible cell mass was observed. After filtering through a 70 ⁇ m filter, centrifuge at 1000 rpm, centrifuge at room temperature, discard the digestive juice, and leave a cell pellet. Finally, the cells were resuspended in RPE cell culture medium (same as in Example 3) to obtain a single cell suspension of RPE, that is, hPSC-derived RPE cells were obtained. The three strains of human pluripotent stem cells (hPSC) of Example 1 were able to obtain RPE cells as described above.
  • TrypLE Express solution Gibco, 12604-013
  • Example 7 Primary culture of hPSC-derived human RPE cells
  • the hPSC-derived RPE single cell suspension prepared according to the method of the present invention was counted using a hemocytometer.
  • the pre-Matrigel-coated six-well culture plate was inoculated at a density of 5 ⁇ 10 4 cells/cm 2 , and primary cell culture was carried out at 37 ° C, 5% CO 2 , and saturated humidity, and the medium was an RPE cell culture medium. After 30 minutes of cell inoculation, the cells began to adhere to the wall, which was round, translucent and refractive, and most contained pigment particles (Fig. 4).
  • the RPE cells prepared by the method were completely fused on the 7th day of primary culture, and were polygonal and filled with pigment (Fig. 5).
  • the primary culture results of RPE cells purified and purified by three human pluripotent stem cells (hPSC) were the same.
  • Example 8 Subculture of hPSC-derived human RPE cells
  • RPE cells (source purchased from Life Technology, USA ( Episomal hiPSC Line, A18945) was cultured for 7-8 days in primary culture and subcultured at 100% cell fusion. The RPE cells to be passaged were taken, the medium was discarded, and the PBS was washed twice. RPE cells was digested with TrypLE TM Express, digested 7-10min 37 °C. The digest was neutralized with RPE cell culture medium (the same RPE cell culture medium as in Example 3). The digested RPE cells were collected and counted. The digest was removed by centrifugation and the cells were resuspended in RPE cell culture medium.
  • the Matrigel-coated plates were seeded at a density of 2-5 x 10 4 cells/cm 2 and subcultured at 37 ° C in a 5% CO 2 incubator.
  • the RPE cells prepared by the method have high purity and strong cell proliferation ability, can be subcultured once a week, can be passaged at least 5 times, and maintain good RPE cell morphology, arranged in a polygonal, cobblestone-like arrangement, containing light pigments (Fig. 6).
  • the pigment particles of the cells gradually decrease as the number of passages increases, similar to the culture characteristics of human embryo RPE cells.
  • RPE cells derived from BC1-GFP-hiPSC and BC1-hiPSC were obtained by the above subculture and amplification methods, and the same results were obtained.
  • Example 9 Cryopreservation and resuscitation culture of hPSC-derived human RPE cells
  • RPE cells (source purchased from Life Technology, USA ( Episomal hiPSC Line, A18945) hiPSC) After growth to fusion, cells were digested by passage of cells of Example 8, and centrifuged for routine cell cryopreservation.
  • the cryopreservation solution was an RPE cell culture medium containing the volume fraction of 10% DMSO (the same as the RPE cell culture medium in Example 3).
  • the cryopreserved RPE cells can be resuscitated, and the cells are cultured under the same conditions as subculture. After resuscitation, the cell proliferative ability is strong, and 100% cell fusion can be achieved in 7-8 days, with the same typical RPE cell characteristics.
  • RPE cells derived from BC1-GFP-hiPSC and BC1-hiPSC were obtained by the above-described cryopreservation and resuscitation culture methods, and the same results were obtained.
  • Example 10 Growth kinetic analysis of hPSC-derived human RPE cells
  • RPE cell growth potential The 5th generation RPE cells obtained by differentiation of BC1-GFP-hiPSC were inoculated into Matrigel-coated plates at a density of 5 ⁇ 10 4 cells/cm 2 at 37 ° C, 5 . % CO 2 , saturated humidity, subcultured once every 7 days, the medium was RPE cell culture medium. Serial passages were passed to passage 10, and the total number of cells obtained per generation was counted using a hemocytometer.
  • the BC1-GFP-RPE cells prepared by the method of the present invention i.e., RPE cells obtained by inducing differentiation by BC1-GFP-hiPSC
  • the cells When the cells were seeded at a Matrigel-coated plate at a cell density of more than 2 ⁇ 10 4 /cm 2 , the cells were grown to have a degree of fusion of more than 90% in about 7 days.
  • the method of the present invention can achieve nearly 15-fold amplification efficiency, and can be amplified more than 3000 times in three consecutive passages.
  • RPE cell growth curve The 5th generation RPE cells obtained by differentiation of BC1-GFP-hiPSC were inoculated into a 96-well plate at a density of 5 ⁇ 10 4 cells/cm 2 , and the medium was RPE cell culture medium, and 3 wells per day were taken. Cell digestion counts for 7 consecutive days. The cell growth curve was traced by the number of cells as the ordinate axis and the number of culture days as the abscissa axis. The doubling days of the RPE cells prepared by the method of the present invention are 1.52 days, and it takes only 7 days to complete the amplification of 1 generation.
  • Example 11 Identification of specific molecular tag expression of hPSC-derived human RPE cells by immunofluorescence method
  • the RPE cells prepared by the method of the present invention were subcultured to a Matrigel-coated coverslip and cultured in an RPE cell culture medium containing 10% FBS. When the cells reached 100% confluence, the FBS was removed and the culture was continued with serum-free RPE cell culture medium.
  • the serum-free RPE cell culture medium is formulated to contain 2 mL of 50 ⁇ B27 nerve cell growth additive (Gibco, 12587-010), 1 mL of 100 ⁇ streptomycin mixture (Gibco, 15240), 1 mL of 100 ⁇ per 100 mL of cell culture medium.
  • Non-essential amino acids (Gibco, 11140-050), 1 mL 100 ⁇ glutamine (Gibco, 35050-061) and 0.1 mL 1000 ⁇ taurine (Sigma, T-0652), balance DMEM/F12 (3:1)
  • the DMEM/F12 (3:1) mixed medium is a mixture of DMEM/F12 (1:1) (Gibco, C11330500BT) and DMEM (Gibco, C11995500BT) in a volume ratio of 3:2.
  • the coverslips were taken at different time points after inoculation culture, washed once with PBS, and fixed on ice for 4-10 minutes with 4% paraformaldehyde.
  • the first antibodies used were: PAX6 (mouse, 1:50, DSHB), OTX2 (rabbit, 1:200, abcam), RPE65 (mouse, 1:500, abcam), ZO-1 (mouse, 1: 400, Life Technology), CHX10 (Sheep, 1:200, Millipore).
  • the RPE cells prepared by the method of the present invention express the characteristic molecular tags PAX6, OTX2, RPE65, ZO-1 (Fig. 7) of RPE cells, and do not express the retinal neural precursor cell tag CHX10.
  • Source purchased from Life Technology, USA The hiPSC and BC1-hiPSC of Episomal hiPSC Line, A18945) induced differentiation and purification of the obtained RPE cells, and the same results were obtained.
  • RPE cells prepared by the method of the present invention were inoculated with some cells to Matrigel pre-coated Transwell (Corning, 0.4 um polyester transparent membrane, The product was cultured in RPE cell culture medium (formulation as in Example 3) containing 10% FBS in Cat. No. 3470). When the cells reached 100% confluence, the FBS was removed for about 7-8 days, and the culture was continued with serum-free RPE cell culture medium (formulation as in Example 11). . The cells were cultured for 6-8 weeks, and their transepithelial electrical resistance (TER) was measured with a transepithelial electrical resistance meter (WPI, EVOM2).
  • TER transepithelial electrical resistance
  • the TER of the Matrigel coated Transwell was measured as the background value.
  • the instrument displays the value minus the background value as the actual TER value.
  • the TER measurement of the RPE per well was repeated after 3 points, and the average of the three groups was repeated.
  • the RPE cell line ARPE-19 was used as a cell control group.
  • the RPE cells (hPSC-RPE) prepared by the method of the present invention can form a good electrical impedance (520.3 ⁇ 23.6 ⁇ *cm 2 ), which is significantly higher than the electrical impedance formed by ARPE-19 cells (210.7 ⁇ 10.5 ⁇ *cm 2 ). (Fig.
  • RPE cells prepared by the method of the present invention have similar electrical impedance resistance functions to RPE cells in vivo, and can form a good electrical resistance difference, which is superior to the ARPE-19 cell line.
  • Source purchased from Life Technology, USA The hiPSC and BC1-hiPSC of Episomal hiPSC Line, A18945) induced differentiation and purification of the obtained RPE cells, and the same results were obtained.
  • PEDF secretion assay RPE cells prepared by the method of the present invention (RPE cells obtained by differentiation and purification from BC1-GFP-hiPSC) were inoculated into Transwell at a density of 5 ⁇ 10 4 cells/cm 2 , and the culture conditions were the same. Epithelial resistance detection section. When TER is greater than 200 ⁇ /cm 2 and the cells are washed 3 times with PBS, the fresh serum-free RPE cell culture medium is replaced (formulation is the same as in Example 11), 120 ⁇ L of Transwell is added to the upper chamber, and 1 mL is added to the lower chamber at 37 ° C, 5% CO 2 .
  • the culture tank under saturated humidity was collected for 24 hours, and the medium of the upper and lower chambers was separately collected.
  • the content of human pigment epithelium-derived factor (PEDF) in the upper and lower chamber cultures of Transwell was determined by ELISA.
  • the ELISA kit was purchased from Wuhan Huamei Bioengineering Co., Ltd. (Cat. No. CSB-EO8818h). Operate according to the experimental method of the manufacturer's instructions.
  • the RPE cells prepared by the method of the invention have the ability to secrete the cytokine PEDF, and the transwell upper chamber PEDF concentration (25.3 ⁇ 3.5 ng/mL) is higher than the lower chamber (7.3 ⁇ 0.8 ng/mL) (Fig. 9), and the body.
  • the function of human RPE cells is similar.
  • Source purchased from Life Technology, USA ( The hiPSC and BC1-hiPSC of Episomal hiPSC Line, A18945) induced differentiation and purification of the obtained RPE cells, and the same results were obtained.
  • the RPE cells prepared by the method of the present invention are subcultured for 7-8 days, and when the cells reach 100% fusion, the serum RPE cell culture medium is replaced with serum-free RPE.
  • the cell culture solution (formulation as in Example 11) was continued for 6-8 weeks.
  • TrypLE TM Express adherent RPE cells was digested to a single cell suspension. Centrifuge at 1000 rpm for 5 min, resuspend in 2 mL of 1% paraformaldehyde solution, and fix the cells for 15 minutes.
  • the cells were washed with PBS containing 0.04% triton-X-100 and 2% sputum serum, and repeated 2 times.
  • the primary antibody RPE65 (mouse, abcam, cat. AB78036) was diluted with PBS containing 0.25% triton-X-100 and 2% sputum serum, and the cells were incubated for one hour at room temperature with a primary antibody concentration of 2 ⁇ g/1 ⁇ 10 6 . Cells. The cells were washed as described above.
  • the anti-mouse Alexa555-labeled secondary antibody (1:500; Life Technologies) was selected and incubated for 30 minutes at room temperature.
  • the PBS was resuspended to 500 ⁇ L and analyzed by a machine.
  • Cell tubes lacking primary antibodies served as parallel negative controls.
  • the flow cytometer is from BD, model LSRFortessa.
  • the RPE cells prepared using the method of the present invention expressed a specific RPE65 molecular tag, and the percentage of positive cells was 98.1% (Fig. 10). It is indicated that the RPE cells prepared by the method of the invention have high purity and can be used for related research.
  • Source purchased from Life Technology, USA ( The hiPSC and BC1-hiPSC of Episomal hiPSC Line, A18945) induced differentiation and purification of the obtained RPE cells, and the same results were obtained.

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Abstract

提供了人多能干细胞来源人视网膜色素上皮细胞的制备和扩增培养方法。该制备方法包括以下步骤：收集人多能干细胞来源的3D-RPE球体，机械分离、去除不含色素的非RPE细胞或团块，留取含有色素的RPE细胞片；酶解消化含有色素的RPE细胞片，获得RPE单细胞悬液，由此获得人多能干细胞来源人视网膜色素上皮细胞。该扩增方法包括将RPE单细胞悬液离心后弃上清再用RPE细胞培养基重悬，接种到预先包被了细胞外基质的细胞培养容器中进行原代培养，细胞长满后进行传代培养。

Description

一种人多能干细胞来源人视网膜色素上皮细胞的制备和扩增培养方法 技术领域：
本发明属于干细胞技术领域，具体涉及一种人多能干细胞来源人视网膜色素上皮细胞的制备和扩增培养方法。
背景技术：
视网膜色素上皮(RPE,retinal pigment epithelium)位于视网膜神经上皮层外侧，为后者提供营养并参与光转导反应。其变性死亡或功能异常是视网膜变性眼病的重要原因。视网膜色素上皮细胞移植是最有前景的复明手段之一，但RPE细胞缺乏制约了该治疗措施的开展。干细胞技术兴起之前，RPE细胞来源局限于从早期流产胚胎或自愿者捐赠的眼球分离获得。最近研究表明，人多能干细胞(human pluripotent stem cell,hPSC)，包括人胚胎干细胞(human embryonic stem cell,hESC)和人诱导多能干细胞(human induced pluripotent stem cell,hiPSC)，具有向视网膜色素上皮细胞分化的能力。hPSCs诱导来源的RPE细胞是治疗视网膜变性疾病最有发展前景的种子细胞。多能干细胞分化为RPE细胞的诱导方法大体分为两类，2D传统贴壁培养法和3D视网膜诱导分化法。然而，这些方法获得的RPE通常与hPSC分化过程中产生的非RPE细胞混杂在一起。因此，如何将其中的RPE细胞分离纯化，获取RPE种子细胞，供相关研究或治疗使用，是目前需要解决的技术难题。
发明内容：
本发明的目的在于克服现有技术中的缺陷，提供一种简单、适用范围广的人多能干细胞来源人视网膜色素上皮细胞的制备和扩增培养方法。
本发明的一种人多能干细胞来源人视网膜色素上皮细胞的制备方法，包括以下步骤：收集人多能干细胞来源的3D-RPE球体，机械分离、去除不含色素的非RPE细胞或团块，留取含有色素的RPE细胞片，酶解消化含有色素的RPE细胞片，获得RPE单细胞悬液，由此获得人多能干细胞来源人视网膜色素上皮细胞。
本发明方法制备的多能干细胞来源RPE细胞的特性与人胚胎来源RPE细胞 特性极其类似，具有典型RPE细胞形态特征，表达特异分子标签PAX6，OTX2，ZO-1和RPE65，并呈现正常生理功能如极性分泌细胞因子PEDF。
所述的人多能干细胞优选为人胚胎干细胞或人诱导多能干细胞。这两种细胞用公知的方法进行细胞培养。
所述的3D-RPE球体是人多能干细胞定向诱导分化为含有RPE细胞的视网膜细胞，随后将贴壁细胞包括RPE细胞刮起，进行悬浮培养，获得3D-RPE球体。3D-RPE球体可以是游离的，也可以粘附于神经视网膜杯的一侧，或粘附于其他细胞团的一侧。
所述的3D-RPE球体优选为人多能干细胞诱导分化40天以上的3D-RPE球体。hPSC启动分化(即hPSC的扩增培养液更换为分化培养液或制备拟胚体)的当天设置为分化的第“0”天。
所述的机械分离、去除不含色素的非RPE细胞或团块，留取含有色素的RPE细胞片具体为：将所有3D-RPE球体转移到细胞容器内，用消化液于37℃水浴下消化8-15min，吸除消化液，1×PBS洗若干次后，使用钨丝针将含有色素的RPE细胞片与无色素的非RPE细胞或团块分离，留取含有色素的RPE细胞片。
所述的消化液优选为质量分数1-2％的Dispase II溶液。
所述的酶解消化含有色素的RPE细胞片，获得RPE单细胞悬液，其步骤具体为：将含有色素的RPE细胞片，转移到TrypLE Express溶液中，37℃水浴下消化7-10min，离心去消化液，用RPE细胞培养基重悬，70-100μm滤网过滤细胞，获得RPE单细胞悬液。
本发明还提供了一种人多能干细胞来源人视网膜色素上皮细胞的扩增培养方法，其特征在于，将上述RPE单细胞悬液离心后弃上清再用RPE细胞培养基重悬，接种到预先包被了细胞外基质的细胞培养容器中进行原代培养，细胞长满后进行传代培养，获得人多能干细胞来源人视网膜色素上皮细胞。
优选，所述的原代培养接种的细胞密度大于等于5×10 4个细胞/cm 2。
所述的细胞长满后进行传代培养具体为：当原代培养的细胞融合度达到90-100％时，弃培养基，PBS洗涤，用TrypLE Express，37℃培养箱内消化7-10min，用RPE细胞培养基终止消化，用移液枪轻轻吹下细胞，离心去除消化液，用RPE细胞培养基重悬细胞，接种细胞到预先包被了细胞外基质的细胞培养容器内，进 行传代培养；当细胞融合度达到90-100％时，重复上述步骤进行反复传代培养；所述的传代培养接种的细胞密度大于等于2×10 4个细胞/cm 2。本发明的方法获得的人RPE细胞至少可以传5代以上。
所述的细胞外基质优选为Matrigel或Gelatin。
所述的细胞培养容器优选为培养板、培养皿或培养瓶。
所述的原代培养和传代培养的细胞培养基的配方均为：每100mL细胞培养基含有10mL胎牛血清、2mL 50×B27、1mL 100×青链霉素混合液、1mL 100×非必需氨基酸、1mL 100×谷氨酰胺和0.1mL 1000×牛磺酸，余量为DMEM/F12(3:1)混合培养基，所述的DMEM/F12(3:1)混合培养基是由DMEM/F12(1:1)培养基和DMEM培养基按照体积比为3:2混合而成。
所述的人RPE细胞冻存，具体为：所获得的RPE原代细胞及传代细胞均可以用公知的方法进行冻存。冻存液为含有体积分数为10％DMSO的细胞培养基。冻存后的RPE细胞可以复苏并具有同样细胞特性。
本发明方法制备的多能干细胞来源人RPE细胞特性与人胚胎来源RPE细胞特性极其类似，具有典型RPE细胞形态特征，表达特异分子标签PAX6，OTX2，ZO-1和RPE65，并呈现正常生理功能如极性分泌细胞因子PEDF，因此，可为相关研究与治疗提供种子细胞材料。
本发明的方法与现有技术相比，具有以下优点：
1.适用范围广，适用于从hPSC制备、纯化、扩增人RPE细胞，包括从人多能干细胞系(hESC和hiPSC)经2D或3D诱导分化条件获得的RPE细胞。
2.技术简便，主要通过机械分离和酶消化来分离、纯化、扩增RPE细胞，不依赖流式细胞仪、磁珠分选或报告基因标记技术等复杂实验设备和技术。消化RPE所用的消化液Dispase II和TrypLE Express也比较温和，对RPE细胞的损害轻微。采用本发明方法，已成功完成三种不同hPSC系来源的RPE细胞的制备，每个细胞系重复了至少3次以上的实验。整套实验技术简便易学，初学者也可快速掌握，成本低，效益好。
3.用本发明方法获得的hPSC来源人RPE细胞具有良好的扩增、传代能力，生长特性良好，产量高，可以批量获取，减少批次间的差异。以大于2×10 4个细胞/cm 2的密度接种于Matrigel包被的培养板时，细胞生长7天左右可有90％以上 融合度。本发明方法制备的RPE细胞经过扩增培养，可获得接近15倍扩增效率，连续传3代可扩增超过3000倍。本发明方法获得的RPE细胞倍增天数约1.52天，完成扩增1代仅需7天，至少可以传代5次以上，并且具有冻存复苏能力。
4.本发明方法制备的hPSC来源人RPE细胞可获得典型RPE细胞形态并色素化，与人胚胎来源RPE细胞的培养特性和细胞形态类似。
5.本发明方法制备的RPE细胞表达RPE特异分子标签PAX6，OTX2，ZO-1，RPE65，与人胚胎视网膜来源RPE细胞特性类似。
6.本发明方法制备的人RPE细胞的功能特性也与体内RPE细胞类似，包括可形成跨上皮电阻和极性分泌PEDF等细胞因子，提示具有良好的应用前景。
7.本发明方法制备的RPE细胞纯度高，达98％，可以作为研究材料广泛应用到相关领域，解决种子细胞瓶颈问题。
综上所述，本发明建立了从hPSC制备人RPE细胞的新技术，可以批量获取人RPE。获得的人RPE细胞在生长特性、细胞形态、特异分子表达谱及功能上均与人胚胎视网膜来源RPE极其类似，并且纯度高，提示用本发明方法制备的RPE在组织工程、再生医学、疾病机制及药物筛选等相关领域具有广阔应用前景，特别是为视网膜疾病研究和治疗提供种子细胞，解决了现有人视网膜色素上皮细胞来源有限、RPE移植供体缺乏的瓶颈问题。本发明方法达同领域世界领先水平，对视网膜疾病患者复明治疗具有重大意义。
附图说明：
图1是hPSC的倒置显微镜图片。
图2是hPSC诱导分化26天后形成的RPE细胞的倒置显微镜图片(40×)。
图3是hPSC诱导分化悬浮培养获得的3D-RPE球体的倒置显微镜图片(40×)。3D RPE球体贴附于神经视网膜(NR)一侧(A)或细胞团块一侧(B)。
图4是3D-RPE球体经过机械分离纯化，并酶解消化获取的单个RPE细胞图片(100×)。
图5是实施例6制备的RPE细胞原代培养七天的照片(200×)。
图6是实施例6制备的RPE细胞，第五次传代培养七天的照片(100×)。
图7是免疫荧光结果照片。
图8是穿上皮电阻(TEER)检测的结果图。
图9是ELISA法检查PEDF的结果图。
图10是流式细胞检测结果。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：人多能干细胞的扩增培养
三株人多能干细胞(hPSC)系用于研究。BC1-GFP-hiPSC及BC1-hiPSC为朋友赠送，另一株hiPSC购自美国Life Technology公司(
Figure PCTCN2018085026-appb-000001
Episomal hiPSC Line,A18945)。所有细胞均接种于细胞外基质MatriGel(Corning,354277)包被了的6孔培养板，用mTeSR1扩增培养。细胞融合度达80％-90％时，用0.5mM EDTA(Life,15575-038)消化，1:8到1:12进行细胞传代培养。倒置显微镜观察，细胞呈片状，集落样生长，集落内细胞排列紧密，边界不清(图1)，由此扩增得到人多能干细胞(hPSC)。
实施例2：hPSC向视网膜细胞包括色素上皮细胞诱导分化
参考已报道的方法(参考文献：Xiufeng Zhong,et al.Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.Nat Commun.2014 Jun 10；5:4047)，诱导hPSC分化为视网膜细胞包括RPE细胞。hPSC启动分化(即hPSC的扩增培养液更换为分化培养液或制备拟胚体)的当天设置为分化的第“0”天。
实施例3：hPSC来源3D-RPE球体的获得及培养
hPSC诱导分化第4周的视网膜细胞包括RPE细胞，显微镜下轻度隆起、环形、折光性强的神经视网膜(NR)和RPE均可辨认。RPE可以围绕NR生长，也可以单独点、片状生长(图2)。用自制的钨丝针或1mL注射器将NR及其附近的RPE挑起，转移到低吸附的培养皿内悬浮培养，培养基为RPE细胞培养基。其配方为：每100mL细胞培养基含有10mL胎牛血清(Gibco,10099-141)、2mL 50×B27神经细胞生长添加剂(Gibco,12587-010)、1mL 100×青链霉素混合液(Gibco,15240)、1mL 100×非必需氨基酸(Gibco,11140-050)、1mL 100×谷氨酰胺(Gibco,35050-061)和0.1mL 1000×牛磺酸(Sigma,T-0652)，余量为DMEM/F12 (3:1)培养基，所述的DMEM/F12(3:1)混合培养基是由DMEM/F12(1:1)(Gibco,C11330500BT)和DMEM(Gibco,C11995500BT)按照体积比为3:2混合而成。剩余细胞在第4周到第6周的任何时间都可用细胞刮刮起，转移到低吸附的培养皿内常规细胞培养箱内悬浮培养，培养基为RPE细胞培养基。悬浮培养的RPE通常卷曲成球(即为3D-RPE球体)，贴在NR一侧或其他细胞团一侧(图3)，也可以游离于培养物中。
实施例4：收集并机械分离纯化hPSC来源3D-RPE球体
hPSC诱导分化6周后，收集悬浮培养的所有3D-RPE球体，包括游离的，粘附于NR或其他细胞团的，置于60mm的培养皿内。解剖显微镜下，用1ml注射器针头细心分解RPE球体，去除NR及非RPE团块，留取含有色素的3D-RPE球体。
实施例5：hPSC来源人RPE片的分离纯化
实施例4纯化出的3D-RPE球体，用质量分数1％的Dispase II(Sigma,D4693-1G)，37℃消化8-15min，去除消化液，1×PBS洗3次。3D-RPE球体表面为RPE，呈黑色，其内部为非RPE细胞团块。解剖显微镜下，使用钨丝针将二者解剖分离，收集分离到的RPE细胞片。
实施例6：hPSC来源人RPE单细胞悬液的制备
用TrypLE Express溶液(Gibco,12604-013)处理分离纯化的RPE细胞片，37℃水浴消化7-10min，加入等体积RPE细胞培养基终止消化，1ml枪头吹打分散细胞，直至无可见的细胞块，70μm滤网过滤后，1000转/分钟，室温离心，弃除消化液，留取细胞沉淀。最后用RPE细胞培养基(同实施例3)重悬细胞，获得RPE单细胞悬液，即得到hPSC来源的RPE细胞。实施例1的三株人多能干细胞(hPSC)按照上述方法都能得到RPE细胞。
实施例7：hPSC来源人RPE细胞的原代培养
按本发明方法制备的hPSC来源RPE单细胞悬液，用血细胞计数板计数细胞总数。以5×10 4个细胞/cm 2的密度接种于预先Matrigel包被的六孔培养板，37℃、5％CO 2，饱和湿度下进行原代细胞培养，培养基为RPE细胞培养基。细胞接种30分钟后，细胞开始贴壁，呈圆形，透亮、富有折光性，多数含有色素颗粒(图4)。用本方法制备的RPE细胞，原代培养第7天，细胞已完全融合，呈多边形， 充满色素(图5)。由三株人多能干细胞(hPSC)分化纯化得到的RPE细胞原代培养结果相同。
实施例8：hPSC来源人RPE细胞的传代培养
RPE细胞(来源购自美国Life Technology公司(
Figure PCTCN2018085026-appb-000002
Episomal hiPSC Line,A18945)的hiPSC)原代培养7-8天，细胞融合度达100％时进行传代培养。取需传代的RPE细胞，弃培养基，PBS洗涤2次。用TrypLE TM Express消化RPE细胞，37℃消化7-10min。用RPE细胞培养基(同实施例3中的RPE细胞培养基)中和消化液。收集消化下来的RPE细胞并计数。离心去除消化液，RPE细胞培养基重悬细胞。以2-5×10 4个细胞/cm 2的密度接种于Matrigel包被的培养板，置于37℃，5％CO 2培养箱进行传代培养。用本方法制备的RPE细胞，纯度高，细胞增殖能力强，每周可传代1次，至少可以传代5次以上，保持良好的RPE细胞形态特征，呈多边形，鹅卵石样排列，含淡淡色素(图6)。细胞的色素颗粒，随着传代次数的增加而逐渐减少，与人胚胎RPE细胞的培养特性类似。另外来源自BC1-GFP-hiPSC及BC1-hiPSC的RPE细胞参照上述传代培养及扩增方法，获得相同的结果。
实施例9：hPSC来源人RPE细胞的冻存与复苏培养
RPE细胞(来源购自美国Life Technology公司(
Figure PCTCN2018085026-appb-000003
Episomal hiPSC Line,A18945)的hiPSC)生长至融合后，用实施例8传代细胞的方法消化细胞，离心后进行常规细胞冻存。冻存液为含有体积分数10％DMSO的RPE细胞培养基(同实施例3中的RPE细胞培养基)。冻存后的RPE细胞可以复苏，用传代培养的同样条件进行细胞培养。复苏后细胞增殖能力强，7-8天即可达到100％细胞融合度，具有同样典型的RPE细胞特征。另外来源自BC1-GFP-hiPSC及BC1-hiPSC的RPE细胞参照上述冻存与复苏培养方法，获得相同的结果。
实施例10：hPSC来源人RPE细胞的生长动力分析
RPE细胞生长势：取BC1-GFP-hiPSC诱导分化获得的第5代RPE细胞，以5×10 4个细胞/cm 2的密度接种到Matrigel包被的培养板培养，培养条件为37℃、5％CO 2、饱和湿度，7天传代一次，培养基是RPE细胞培养基。连续传代至第10代，使用血细胞计数板计数每代获取的细胞总量。用本发明方法制备的BC1-GFP-RPE细胞(即BC1-GFP-hiPSC诱导分化获得的RPE细胞)可以稳定 扩增，连续传代5代以上。以大于2×10 4/cm 2的细胞密度接种于Matrigel包被的培养板时，细胞生长7天左右可有90％以上融合度。本发明的方法可获得接近15倍扩增效率，连续传3代可扩增超过3000倍。
RPE细胞生长曲线：取BC1-GFP-hiPSC诱导分化获得的第5代RPE细胞，5×10 4个细胞/cm 2的密度接种于96孔板，培养基是RPE细胞培养基，每天取3孔细胞消化计数，连续7天。以细胞数量为纵坐标轴，培养天数为横坐标轴，描记细胞生长曲线。本发明方法制备的RPE细胞倍增天数为1.52天，完成扩增1代仅需7天。
实施例11：免疫荧光方法鉴定hPSC来源人RPE细胞的特异分子标签表达情况
用本发明方法制备的RPE细胞(来源BC1-GFP-hiPSC诱导分化获得的RPE细胞)，传代时接种部分细胞到Matrigel包被的盖玻片上，用含有10％FBS的RPE细胞培养基培养。细胞达到100％融合时，将FBS去除，用无血清的RPE细胞培养液继续培养。无血清的RPE细胞培养基配方为：每100mL细胞培养基含有2mL 50×B27神经细胞生长添加剂(Gibco,12587-010)、1mL 100×青链霉素混合液(Gibco,15240)、1mL 100×非必需氨基酸(Gibco,11140-050)、1mL 100×谷氨酰胺(Gibco,35050-061)和0.1mL 1000×牛磺酸(Sigma,T-0652)，余量为DMEM/F12(3:1)培养基，所述的DMEM/F12(3:1)混合培养基是由DMEM/F12(1:1)(Gibco,C11330500BT)和DMEM(Gibco,C11995500BT)按照体积比为3:2混合而成。接种培养后不同时间点取出盖玻片，PBS洗1次后，用4％多聚甲醛在冰上固定5-10分钟。PBS洗三次后，加入封闭液(10％正常驴血清和0.25％Triton X-100的PBS)在室温封闭1小时。随后，4℃过夜孵育一抗。所用第一抗体为：PAX6(小鼠，1:50，DSHB)，OTX2(兔，1:200，abcam)，RPE65(小鼠，1:500，abcam)，ZO-1(小鼠，1:400，Life Technology)，CHX10(绵羊，1:200，Millipore)。次日，PBS洗涤细胞3次，用荧光素标记的第二抗体孵育细胞(1:500；Life Technologies)，室温1小时。二抗孵育后，PBS洗涤细胞，DAPI染色10分钟。PBS洗涤3次后，Olympus荧光显微镜观察、拍照。本发明方法制备的RPE细胞表达RPE细胞特征性分子标签PAX6，OTX2，RPE65，ZO-1(图7)，不表达视网膜神经前体细胞标签CHX10。来源购自美国Life Technology 公司(
Figure PCTCN2018085026-appb-000004
Episomal hiPSC Line,A18945)的hiPSC和BC1-hiPSC诱导分化纯化得到的RPE细胞，获得相同的结果。
实施例12：本发明方法制备的RPE细胞的功能分析
跨上皮电阻检测：用本发明方法制备的RPE细胞(来源BC1-GFP-hiPSC诱导分化获得的RPE细胞)，传代时接种部分细胞到Matrigel预包被的Transwell(Corning，0.4um聚酯透明膜，货号3470)内，用含有10％FBS的RPE细胞培养基(配方同实施例3)培养。细胞达到100％融合时，约7-8天，将FBS去除，用无血清的RPE细胞培养基(配方同实施例11)继续培养。。细胞接种培养6-8周，用跨上皮电阻仪(WPI,EVOM2)测量其跨上皮电阻(TER)。电极用75％酒精浸泡消毒后，Hank’s平衡盐溶液平衡后再使用。测量Matrigel包被的Transwell的TER作为背景值。仪器显示数值减去背景值为实际TER值。每孔RPE的TER测量要重复3个点后取平均值，重复3组实验。RPE细胞系ARPE-19作为细胞对照组。用本发明方法制备的RPE细胞(hPSC-RPE)可以形成良好的电阻抗(520.3±23.6Ω*cm 2)，明显高于ARPE-19细胞所形成的电阻抗(210.7±10.5Ω*cm 2)(图8)，提示用本发明方法制备的RPE细胞与体内RPE细胞具有类似的电阻抗功能，可以形成良好的电阻差，优于ARPE-19细胞系。来源购自美国Life Technology公司(
Figure PCTCN2018085026-appb-000005
Episomal hiPSC Line,A18945)的hiPSC和BC1-hiPSC诱导分化纯化得到的RPE细胞，获得相同的结果。
PEDF分泌测定：用本发明方法制备的RPE细胞(来源BC1-GFP-hiPSC诱导分化纯化获得的RPE细胞)，以5×10 4个细胞/cm 2的密度接种于Transwell中培养，培养条件同跨上皮电阻检测部分。当TER大于200Ω/cm 2，PBS洗涤细胞3次后，更换新鲜无血清RPE细胞培养液(配方同实施例11)，Transwell上室加入120μL，下室加入1mL，于37℃、5％CO 2，饱和湿度下的培养箱，培养24小时后，分别收集上下小室的培养基。使用ELISA法检测Transwell上下小室培养液的人色素上皮衍生因子(PEDF)的含量。ELISA试剂盒购于武汉华美生物工程有限公司(货号CSB-EO8818h)。按照制造商说明书的实验方法操作。本发明方法制备的RPE细胞具有极性分泌细胞因子PEDF的能力，Transwell上室PEDF浓度(25.3±3.5ng/mL)高于下室的(7.3±0.8ng/mL)(图9)，与体内人RPE细胞的功能类似。来源购自美国Life Technology公司(
Figure PCTCN2018085026-appb-000006
Episomal  hiPSC Line,A18945)的hiPSC和BC1-hiPSC诱导分化纯化得到的RPE细胞，获得相同的结果。
实施例13：细胞流式技术分析本方法制备的RPE细胞纯度
本发明方法制备的RPE细胞(来源BC1-GFP-hiPSC诱导分化获得的RPE细胞)，传代培养7-8天，细胞达到100％融合时，将有血清的RPE细胞培养液更换为无血清的RPE细胞培养液(配方同实施例11)，继续培养6-8周。用TrypLE TMExpress将贴壁RPE细胞消化为单细胞悬液。1000rpm离心5min，2mL 1％多聚甲醛溶液重悬，固定细胞15分钟。1000rpm离心5min，用含0.04％triton-X-100和2％驴血清的PBS洗涤细胞，重复2遍。使用含0.25％triton-X-100和2％驴血清的PBS稀释一抗RPE65(小鼠，abcam,cat.AB78036)，细胞室温下孵育一抗1小时，一抗浓度为2μg/1×10 6个细胞。按前述方法洗细胞。选择驴抗鼠Alexa555标记的二抗(1:500；Life Technologies)室温下孵育30分钟。洗细胞后，PBS重悬至500μL，上机分析。缺乏一抗的细胞管作为平行阴性对照。流式细胞分析仪来自BD公司，型号LSRFortessa。使用本发明方法制备的RPE细胞表达特异RPE65分子标签，阳性细胞所占百分比为98.1％(图10)。说明用本发明方法制备的RPE细胞纯度高，可供相关研究使用。来源购自美国Life Technology公司(
Figure PCTCN2018085026-appb-000007
Episomal hiPSC Line,A18945)的hiPSC和BC1-hiPSC诱导分化纯化得到的RPE细胞，获得相同的结果。

Claims (9)

  1. 一种人多能干细胞来源人视网膜色素上皮细胞的制备方法，其特征在于，包括以下步骤：收集人多能干细胞来源的3D-RPE球体，机械分离、去除不含色素的非RPE细胞或团块，留取含有色素的RPE细胞片，酶解消化含有色素的RPE细胞片，获得RPE单细胞悬液，由此获得人多能干细胞来源人视网膜色素上皮细胞。
  2. 根据权利要求1所述的制备方法，其特征在于，所述的3D-RPE球体是将人多能干细胞定向诱导分化为包括RPE细胞的视网膜细胞，随后将贴壁细胞包括RPE细胞刮起，进行悬浮培养，获得3D-RPE球体，3D-RPE球体是游离的，粘附于神经视网膜杯的一侧或粘附于其他细胞团的一侧。
  3. 根据权利要求1所述的制备方法，其特征在于，所述的机械分离、去除不含色素的非RPE细胞或团块，留取含有色素的RPE细胞片具体为：将所有3D-RPE球体转移到细胞容器内，用消化液于37℃水浴下消化8-15min，吸除消化液，1×PBS洗若干次后，使用钨丝针将含有色素的RPE细胞片与无色素的非RPE细胞或团块分离，留取含有色素的RPE细胞片。
  4. 根据权利要求1所述的制备方法，其特征在于，所述的酶解消化含有色素的RPE细胞片，获得RPE单细胞悬液，其步骤具体为：将含有色素的RPE细胞片，用TrypLE Express溶液，37℃水浴消化7-10min，终止消化，70-100μm滤网过滤细胞，离心去消化液，用RPE细胞培养基重悬，获得RPE单细胞悬液。
  5. 一种人多能干细胞来源人视网膜色素上皮细胞的扩增培养方法，其特征在于，将权利要求1的RPE单细胞悬液离心后弃上清再用RPE细胞培养基重悬，接种到预先包被了细胞外基质的细胞培养容器中进行原代培养，细胞长满后进行传代培养，获得人多能干细胞来源人视网膜色素上皮细胞。
  6. 根据权利要求5所述的扩增培养方法，其特征在于，所述的原代培养接种的细胞密度大于等于5×10 4个细胞/cm 2。
  7. 根据权利要求5所述的扩增培养方法，其特征在于，所述的细胞长满后进行传代培养具体为：当原代培养的细胞融合度达到90-100％时，弃培养基，PBS洗涤，用TrypLE Express，37℃培养箱内消化7-10min，用RPE细胞培养基 终止消化，用移液枪轻轻吹下细胞，离心去除消化液，用RPE细胞培养基重悬细胞，接种细胞到预先包被了细胞外基质的细胞培养容器内，进行传代培养；当细胞融合度达到90-100％时，重复上述步骤进行反复传代培养；所述的传代培养接种的细胞密度大于等于2×10 4个细胞/cm 2。
  8. 根据权利要求5所述的扩增培养方法，其特征在于，所述的细胞外基质为Matrigel或Gelatin。
  9. 根据权利要求5所述的扩增培养方法，其特征在于，所述的原代培养和传代培养的RPE细胞培养基的配方均为：每100mL细胞培养基含有10mL胎牛血清、2mL 50×B27神经细胞生长添加剂、1mL 100×青链霉素混合液、1mL 100×非必需氨基酸、1mL 100×谷氨酰胺和0.1mL 1000×牛磺酸，余量为DMEM/F12混合培养基，所述的DMEM/F12混合培养基是由DMEM/F12(1:1)培养基和DMEM培养基按照体积比为3:2混合而成。
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