CN106609257B

CN106609257B - Method for efficiently separating in-vitro induced RPE (RPC-like protein) cells and RPC (RPC-like protein)

Info

Publication number: CN106609257B
Application number: CN201510690678.1A
Authority: CN
Inventors: 徐国彤; 李宗义; 高芙蓉; 吕立夏
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2015-10-22
Filing date: 2015-10-22
Publication date: 2020-04-10
Anticipated expiration: 2035-10-22
Also published as: CN106609257A

Abstract

The invention relates to a method for efficiently separating in vitro induced RPE cells and RPC, belonging to the technical field of biology. The method can simply and effectively separate and purify the mixed cell population from hESCs to obtain a large amount of mature RPE cells and RPC. Compared with the prior art, the invention is suitable for mixed cells containing pigment agglomerates from hESCs; compared with the traditional mechanical separation method, the method disclosed by the invention is simple, good in repeatability, high in success rate, high in cell purity and yield, and capable of separating the RPE cells and the RPC simultaneously.

Description

Method for efficiently separating in-vitro induced RPE (RPC-like protein) cells and RPC (RPC-like protein)

Technical Field

The invention relates to the technical field of biology, in particular to a method for efficiently separating in-vitro induced RPE cells and RPC.

Background

Retinal degenerative diseases are the leading cause of blindness worldwide. In this disease, damage and dysfunction of the Retinal Pigment Epithelium (RPE) leads to degeneration of the photoreceptor cells, ultimately affecting the patient's vision until blindness. Due to the non-regenerative nature of RPE and photoreceptor cells, there is a clinical lack of effective therapeutic measures for such diseases to date. Cell therapy has much greater promise for the treatment of retinal degenerative diseases than other therapies.

Embryonic Stem Cells (ESCs) can be an ideal source of transplanted cells because of their ability to proliferate indefinitely and differentiate into various cells. The feasibility and safety of ESCs-RPE in clinical studies on human retinal degenerative diseases have been reported, although the postoperative vision of patients is far from the requirements of normal life. Therefore, a set of stable and effective methods for differentiating the in vitro totipotent stem cells into the RPE cells is particularly important for the treatment and pharmaceutical research of retinal degenerative diseases.

During the past decade, various methods have been reported to control embryonic stem cell differentiation to retinal pigment epithelial cells and to exert protective effects in various animal models of disease. However, most of these reported methods form a mixed cell population containing pigment aggregates at a later stage, and the cells are generally separated by a mechanical picking method. The method has the problems of long time consumption, large batch-to-batch difference, low cell yield and the like.

Therefore, a method for separating RPE cells and retinal neuron precursor cells (RPCs) induced in vitro is needed, which can simultaneously obtain mature RPE cells and RPC cells, and has the advantages of simple method, fast time, high cell yield, and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for efficiently separating in vitro induced RPE cells and RPC.

The purpose of the invention can be realized by the following technical scheme:

a method for efficiently separating in vitro induced retinal pigment epithelial cells (RPE) and retinal neuron precursor cells (RPC), comprising the following steps:

a. performing enzyme treatment on mixed cells with pigment aggregates from human embryonic stem cells (hESCs) to form single cells, centrifuging, removing supernatant, adding culture medium to suspend, and inoculating to a culture dish for suspension culture;

b. culturing for 1 day to form black and white cell suspension ball, periodically changing liquid and culturing;

c. culturing the mixed cell suspension spheres for 7 days to form a black part and a white part, and then mechanically separating to obtain two types of cell suspension spheres;

d. collecting different cell suspension balls, respectively inoculating the suspension balls into a culture dish, placing for culture, and periodically changing the culture solution to obtain mature RPE cells and RPC.

Preferably, the mixed cells with pigment agglomerates from the hESCs in the step a are obtained by a method of inducing differentiation or spontaneous differentiation of hESCs, the enzyme treatment to form single cells is to use 0.25% pancreatin to treat the single cells for 20 minutes at 37 ℃, blow and beat the single cells by a pipette and then stop pancreatin reaction, the supernatant is discarded after centrifugation is 5 minutes by a horizontal centrifuge at 1800rpm, the culture medium comprises DMEM/F12 basal culture medium, serum substitute, unnecessary amino acid, penicillin, streptomycin, glutamine and β -mercaptoethanol, wherein the volume fraction of the serum substitute is 20%,the concentration of the optional amino acid was 0.01mmol/L, the concentration of penicillin was 100U/ml, the concentration of streptomycin was 100ug/ml, the concentration of glutamine was 1mmol/L, the concentration of β -mercaptoethanol was 0.1mmol/L, the culture was performed at 37 ℃ and 5% CO₂The culture is carried out in an incubator; the culture dish is a non-adhesive bacterial culture dish.

Preferably, the regular solution change in the step b is a daily solution change, the cell suspension balls are collected to a 15 ml centrifuge tube, a horizontal centrifuge is used for centrifuging for 3 minutes at 400rpm, and the supernatant is discarded and added into a fresh culture medium; the culture was at 37 ℃ and 5% CO₂In an incubator.

Preferably, the black and white cells in the mixed suspension ball are respectively aggregated and separated to form two adhered black suspension balls and white suspension balls in the step c; the culture was at 37 ℃ and 5% CO₂The culture is carried out in an incubator; the mechanical separation is to separate the two suspension balls by using a sterilized blade to cut the black and white suspension balls in an adhesion manner.

Preferably, the collecting of different cell suspension spheres in the step d is to collect black and white cell suspension spheres according to colors by using a liquid transfer device; the culture dish is a cell culture dish coated by 100ug/ml Matrigel; the regular liquid change is every other day liquid change; the mature RPE cells have cobblestone-like morphology, accumulated pigments, expressed related proteins and phagocytic function; the culture was at 37 ℃ and 5% CO₂The culture is carried out in an incubator; the RPC is a neuron cell expressing related protein.

The invention provides a method for separating in vitro induced RPE cells and RPC, which can simply and effectively separate and purify a large amount of mature RPE cells and RPC from a mixed cell population derived from hESCs. Compared with the prior art, the invention is suitable for mixed cells containing pigment agglomerates from hESCs; compared with the traditional mechanical separation method, the method disclosed by the invention is simple, good in repeatability, high in success rate, high in cell purity and yield, and capable of separating the RPE cells and the RPC simultaneously.

Drawings

FIG. 1 is a schematic diagram of suspension culture of pigment-containing mixed differentiated cells derived from an embryonic stem cell line H1 of the present invention after enzyme treatment;

FIG. 2 is a schematic illustration of the mechanical separation and collection of black and white cell suspension spheres of the present invention;

FIG. 3 is a schematic diagram showing the morphology of cells in which black and white suspension spheres are attached to the wall and cultured for a long time according to the present invention;

FIG. 4 is a schematic diagram of the morphology and identification of H1-derived RPE cells isolated and purified according to the present invention;

FIG. 5 is a schematic diagram of immunofluorescence assay of isolated and purified RPC derived from H1 according to the present invention.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Examples

This example is a method for efficiently separating retinal pigment epithelial cells and retinal neuron precursor cells induced in vitro.

In this example, the mixed cells are mixed cell populations containing pigment aggregates obtained by differentiation of multi-stem cells, and the separation can be achieved by the method.

In this embodiment, the method for separating cells comprises the following steps:

a. performing enzyme treatment on mixed cells with pigment agglomerates from hESCs to form single cells, centrifuging, removing supernatant, adding a culture medium to perform suspension, and inoculating the suspension in a culture dish for suspension culture;

the hESCs in the step are H1 cell line of 50 th generation, the mixed cells with pigment lumps are obtained by directional induced differentiation of hESCs for 45 days, as shown in figure 1A, the enzyme treatment of the single cells is to apply 0.25% pancreatin to treat the single cells for 20 minutes at 37 ℃, blow the single cells by a pipette and stop the pancreatin reaction, the supernatant after centrifugation is to use a horizontal centrifuge to centrifuge for 5 minutes at 1800rpm and then discard the supernatant, the components of the culture medium are DMEM/F12 basal medium, serum substitute, nonessential amino acid, penicillin, streptomycin, glutamine and β -mercaptoethanol, wherein the volume fraction of the serum substitute is 20%, the concentration of the nonessential amino acid is 0.01mmol/L, the concentration of the penicillin is 100U/ml, and the concentration of the streptomycin is 100U/mlg/ml, glutamine concentration of 1mmol/L, β -mercaptoethanol concentration of 0.1mmol/L, and culturing at 37 deg.C and 5% CO₂The culture is carried out in an incubator; the culture dish is a non-adhesive bacterial culture dish.

the cell suspension of the mixture of black and white cells formed after the day in this step is shown in FIG. 1B; the regular solution changing is that the solution is changed every day, cell suspension balls are collected to 15 ml centrifuge tubes, a horizontal centrifuge is used for centrifuging for 3 minutes at 400rpm, and the supernatant is discarded and added into a fresh culture medium; the culture was at 37 ℃ and 5% CO₂In an incubator.

the black and white parts formed in the step are cell suspension states of 3 days and 7 days as shown in fig. 1C and 1D, wherein black and white cells in the mixed suspension spheres are respectively aggregated and separated to form two adhered black suspension spheres and white suspension spheres; the culture was at 37 ℃ and 5% CO₂The culture is carried out in an incubator; the mechanical separation is that a sterilized blade is used to cut the black and white suspension ball to separate the two suspension balls as shown in fig. 2A and 2B.

In the step, collecting different cell suspension balls is to collect black and white cell suspension balls according to colors by using a liquid transfer device as shown in figures 2C and 2D; the culture dish is a cell culture dish coated by 100ug/ml Matrigel; the regular liquid change is every other day liquid change; the mature retinal pigment epithelial cells are cells with cobblestone-like morphology, pigment accumulation, expression of related proteins and phagocytic function, and as shown in fig. 3A and 3C, are cell morphology diagrams of black cell suspension adherence for 1 week and 3 weeks; the culture was at 37 ℃ and 5% CO₂The culture is carried out in an incubator; the retinal neuron precursor cells are neuron cells expressing related proteins, and as shown in fig. 3B and 3D, the cell morphology of white cell suspension spheres attached for 1 week and 3 weeks.

Further immunohistochemical identification was carried out on the isolated and purified H1-derived RPE cells (H1-RPE), and H1-RPE cells were found to have typical cobblestone-like morphology and pigment accumulation; expresses the mature RPE cell-associated protein ZO-1 and has the ability to phagocytose outer segments, as shown in FIG. 4. Immunohistochemical identification of the isolated and purified H1-derived retinal neuron precursor cells revealed that white suspensor spheres expressed the retinal neuron-associated protein GFAP (glial neuron marker), Rho, Crx and Recoverin (rod marker) after 4 weeks of adherent culture, as shown in FIG. 5.

Finally, the foregoing description of the embodiments is presented to enable a person of ordinary skill in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. A method for efficiently separating in vitro induced RPE cells and RPC cells is characterized by comprising the following steps:

a. carrying out enzyme treatment on mixed cells with pigment agglomerates from human embryonic stem cells to form single cells, centrifuging, removing supernatant, adding a culture medium to suspend, and inoculating the suspension in a culture dish for suspension culture;

d. collecting different cell suspension balls, respectively inoculating the suspension balls into a culture dish, placing for culture, and periodically changing the culture solution to respectively obtain mature RPE cells and RPC cells;

the mixed cells with pigment agglomerates from the human embryonic stem cells in the step a are obtained by a method of induced differentiation or spontaneous differentiation of the human embryonic stem cells;

the step a, treating the enzyme into single cells by using 0.25 percent of pancreatin at 37 ℃ for 20 minutes, blowing the single cells by using a pipette, and then stopping the pancreatin reaction;

the components of the culture medium in the step a are DMEM/F12 basal culture medium, serum substitute, non-essential amino acid, penicillin, streptomycin, glutamine and β -mercaptoethanol, wherein the volume fraction of the serum substitute is 20%, the concentration of the non-essential amino acid is 0.01mmol/L, the concentration of the penicillin is 100U/mL, the concentration of the streptomycin is 100 mu g/mL, the concentration of the glutamine is 1mmol/L, and the concentration of β -mercaptoethanol is 0.1 mmol/L;

the regular liquid change in the step b is that liquid is changed every day, cell suspension balls are collected to 15 ml centrifuge tubes, a horizontal centrifuge is used for centrifuging for 3 minutes at 400rpm, and the supernatant is discarded and added into a fresh culture medium;

d, the mature RPE cells are cobblestone-like cells which have accumulated pigments, express related proteins and have phagocytosis function; the RPC cells are neuron cells expressing related proteins;

the culture dish in the step d is a cell culture dish coated by 100 mu g/mL Matrigel; the regular liquid change is every other day.

2. The method of claim 1, wherein the step c of forming black and white parts comprises aggregating black and white cells in a suspension ball and separating to form two adherent black and white suspension balls; the mechanical separation is to cut the black and white suspension balls at the adhesion part by using a sterilized blade to separate the two suspension balls.

3. The method for efficiently separating in vitro induced RPE cells and RPC cells according to claim 1, wherein the collection of different cell suspension balls in step d is a color collection of black and white cell suspension balls by using a pipette.

4. The method of claim 1, wherein the culturing is at 37 ℃ and 5% CO in each step₂In an incubator.