CN114807034A - Preparation method of Muller cells derived from human pluripotent stem cells - Google Patents
Preparation method of Muller cells derived from human pluripotent stem cells Download PDFInfo
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Abstract
The invention discloses a preparation method of Muller cells derived from human pluripotent stem cells, and belongs to the technical field of cell biology. The method comprises the following steps: inducing and differentiating the human pluripotent stem cells to obtain three-dimensional retina organs; mechanically separating retina organs to obtain a neural retina sheet; digesting the obtained neural retina slices to obtain a retina single cell suspension. And carrying out adherent culture on the obtained retina single cell suspension in Muller cell culture medium, removing non-Muller cells to obtain human Muller cells derived from human pluripotent stem cells, and carrying out subculture expansion and cryopreservation. The human Muller cells obtained by the method make up the limitation of human tissue separation of Muller cells; through identification, the Muller cells obtained by the method do not have the pollution of other retina cells, astrocytes and other non-Muller cells, and have high purity; muller cells derived from retinal organoids at a late stage of induced differentiation, e.g., after day 120, can be expanded continuously for more than 10 generations, can produce a large number of cells in a short period, and can be cryopreserved by a simple method, thereby providing useful cell tools or seed cells for the study of the characteristics and functions of human Muller cells in physiological and pathological states, the study of retinal disease damage repair, and the like.
Description
The technical field is as follows:
the invention belongs to the technical field of cell biology, and particularly relates to a preparation method of Muller cells derived from human pluripotent stem cells.
Background art:
muller cells are the major glial cells of vertebrate retinas and play important physiological functions in the retina, including the formation of the blood retinal barrier, maintaining retinal integrity, etc. They also provide neurotrophic, metabolic and antioxidant support to retinal neurons and regulate neuronal activity through neurotransmitter circulation, with neuroprotective effects (brinkmann et al, 2009; Eastlake et al, 2020; Reichenbach and brinkmann, 2013).
Currently, studies in several lower animal models have shown that under pathological conditions muller cells can dedifferentiate to form retinal precursor-like cells with the potential to differentiate to obtain other retinal cell types, complementing neurons damaged under pathological conditions (Raymond et al, 2006). Thus, over a decade scientists have endeavored to explore the regenerative potential of muller cells and their mechanisms, and it is expected that muller cells will be used to develop new strategies for the treatment of advanced stage neuroretinopathy. It has been found that M uller cells are activated by various pathogenic stimuli (e.g. hypoxia) in various types of human retinal diseases, such as diabetic retinopathy and retinal degenerative diseases (Bringmann et al, 2006). Slight activation of Muller cells may promote the survival of retinal neurons and thus repair the retina. But continued activation may also lead to the formation of reactive gliosis, glial scarring, accelerated retinal neurodegeneration, and severely hamper retinal repair. However, the problems of regeneration ability, regeneration-related mechanism, ability to repair damaged retina, and transformation of clinical treatment in human retina and muller cells are still unclear, and a great deal of preclinical research is urgently needed.
However, since it is difficult for humans to perform in vivo experiments like animal models, the progress of human muller cell-related studies has been largely hampered by past observations of clinical patients. And human muller cells are difficult to obtain in large quantities due to the limited source of human tissue cells, and the physiological characteristics of muller cells are still not well understood. Furthermore, to date, human Muller cells have been isolated primarily from human cadaver donors and specimens remaining after vitreoretinal surgery (Limb et al, 2002; Lawrence et al, 2007). However, in adult retinas, astrocytes, which share many features with muller cells, are also present in the retinal nerve fiber layer, interfering with the expansion and purification of muller cells (Lawrence et al, 2007). In addition, Muller cells derived from human donor tissue are prone to immune rejection and risk of disease transmission, and are not suitable for future clinical applications (Eastlake et al, 2019).
The rapid development of human pluripotent stem cells has made it possible to solve the above problems. Scientists have generated three-dimensional retinal organoids from human pluripotent stem cells that are capable of reproducing human retinal development and forming retinal tissue containing all retinal cell types (Zhong et al, 2014; Li et al, 2018; Guan et al, 2021). Studies have shown that Muller cells are the only glial cell type sharing common retinal precursor cells with retinal neurons, and that they can appear and survive in human pluripotent stem cell-derived retinal organoids (Zhong et al, 2014; Fligor et al, 2018; Capowski et al, 2019; Singh et al, 2021).
The invention content is as follows:
as Muller cells in human retina have a plurality of important functions, the invention aims to provide a preparation and amplification culture method of Muller cells from human pluripotent stem cells, and provide abundant seed cells for the research of pathophysiological characteristics and mechanisms of Muller cells, the research and treatment of Muller cell related diseases and the like.
The invention aims to provide a preparation method of Muller cells derived from human pluripotent stem cells, which comprises the following steps:
a. inducing and differentiating the human pluripotent stem cells to obtain three-dimensional retina organs;
b. removing non-retina organoid tissues, retinal pigment epithelial cells and non-neural retina layers from the three-dimensional retina organoids obtained in the step a to obtain neural retina sheets;
c. b, digesting the neural retina sheet obtained in the step b to obtain a retina single cell suspension;
d. and d, performing adherent culture on the retinal single cell suspension obtained in the step c, removing non-Muller cells to obtain Muller cells obtained by inducing human pluripotent stem cells, and performing passage expansion and cryopreservation.
Preferably, the three-dimensional retina organoid is a three-dimensional retina organoid derived from human pluripotent stem cells induced to differentiate into a late stage, wherein the late stage is after 90-120 days; more preferably, the advanced stage is 120 days later; most preferably, the late phase is after day 150.
Preferably, for step a, human pluripotent stem cells are firstly digested into small cell clusters, placed in mTeSR1 culture medium containing 10 mu M (-) -Blebbistatin to form embryoid bodies, gradually replaced by nerve induction culture medium, and then the embryoid bodies are inoculated into a culture dish coated by Matrigel for adherent culture; differentiating to 16 days, culturing with a retinal differentiation culture medium until retinal tissues protruding out of the bottom surface of the culture dish are formed, picking up the retinal tissues, inoculating the retinal tissues into the retinal culture medium 1, and performing suspension culture to obtain the three-dimensional retinal organoids derived from the human pluripotent stem cells; from day 90 of differentiation, the medium was changed to retina medium 2 and cultured in suspension for a long period.
More preferably, the nerve induction culture medium is heparin which is prepared by adding 1 volume fraction of N2 additive, 1 volume fraction of non-essential amino acid and final concentration of 2 mu g/mL into DMEM/F12 basic culture medium.
More preferably, the retina differentiation medium is a DMEM basal medium with 35% volume fraction, 2% volume fraction B27 additive without vitamin A, 1% volume fraction antibacterial fungicide and 1% volume fraction non-essential amino acid added into a DMEM/F12 basal medium.
More preferably, the retina culture medium 1 is a DMEM/F12 basal medium supplemented with 40% by volume of DMEM basal medium, 2% by volume of B27 additive without vitamin a, 1% by volume of antibacterial fungicide, 1% by volume of nonessential amino acids, 10% by volume of fetal bovine serum, 1% by volume of GlutaMAX, and a final concentration of 100 μ M taurine.
More preferably, the retina culture medium 2 is prepared by adding 1% by volume of N2 additive, 1% by volume of antibacterial fungicide, 1% by volume of nonessential amino acid, 10% by volume of fetal bovine serum, 1% by volume of GlutaMAX and 100 μ M of taurine into a DMEM/F12 basic culture medium.
More preferably, the forming of the embryoid body is to culture the human pluripotent stem cells in mTeSR1 culture medium in an adherent way, when the clone grows to occupy 80-90% of the area of the bottom of the hole, scrape the differentiated cells, suck the mTeSR1 culture medium, rinse the cells with sterile PBS, add 0.5mM EDTA solution, digest the cells for 3-7min at 37 ℃, suck the EDTA, take the mTeSR1 culture medium containing 10 mu M (-) -Blebbistatin to blow down the cells lightly to obtain small cell clusters, and place the small cell clusters in the mTeSR1 culture medium containing 10 mu M (-) -Blebbistatin to form the embryoid body through suspension culture.
More preferably, the step-by-step replacement is carried out by taking the day of establishing the embryoid body as the day of induced differentiation 0, the next day as the day of induced differentiation 1, and so on; the embryoid bodies were inoculated into mTeSR1 medium containing 10. mu.M (-) -Blebbistatin on day 0 for suspension culture, medium mTeSR1 medium containing 10. mu.M (-) -Blebbistatin was replaced with medium A (medium for neural induction mixed with mTeSR1 at 1:3 volume ratio) on day 1, medium A was replaced with medium B (medium for neural induction mixed with mTeSR1 at 1:1 volume ratio) on day 2, and medium B was replaced with medium for neural induction on day 3.
More preferably, the retinal tissue is picked up by drawing a circle around the target retinal tissue, gently picking up the cell layer around the retinal tissue from one side, and completely suspending the retinal tissue in the retinal culture medium 1 without damaging the original structural form of the retinal tissue.
Preferably, in the step b, the three-dimensional retina organoid obtained by differentiation in the step a is placed in a low adsorption culture dish containing a retina culture medium 2, and other non-retina organoid tissues and retinal pigment epithelial cells are mechanically separated and discarded; and then mechanically separating along the junction between the neural retina layer and the non-neural retina layer, and discarding the non-neural retina layer to obtain the neural retina sheet.
Preferably, in the step c, the neural retina patch suspension obtained in the step b is transferred to a centrifuge tube, naturally stands for precipitation, PBS is added for washing after absorbing and removing the culture medium, papain is used after absorbing and removing the PBS, and cells are digested in water bath at 37 ℃ for 30-90min to obtain the retina single cell suspension.
Preferably, in step d, the single retinal cell suspension obtained is seeded on Matrigel-coated cell culture plates containing muller cell culture medium, and the cells are recorded as passage 0; and performing amplification culture on the surviving cells to obtain Muller cells induced by the human pluripotent stem cells.
More preferably, the adherent culture is performed to remove non-Muller cells and obtain Muller cells induced by human pluripotent stem cells, and the obtained retinal single cell suspension is used as>1×10 4 Individual cell/cm 2 Was seeded on Matrigel-coated cell culture plates containing muller cell culture medium and these cells were recorded as passage 0.
More preferably, said passage expansion: and (3) carrying out passage when the Muller cells obtained by the induction of the human pluripotent stem cells grow to reach the fusion degree of 80-90%. Further, specifically, removing culture medium supernatant, washing with PBS, adding TRYPSIN 0.25% EDTA, digesting at 37 deg.C for 5-10min to obtain cell suspension, and mixing the cell suspension with the above extract>1×10 4 Individual cell/cm 2 Is densely inoculated inMatrigel coated cell culture plates of Muller cell culture medium.
More preferably, the cryopreservation: the resulting Muller cell suspension from the digestion is adjusted to a concentration of 0.5-1X 10 6 Dripping equal volume of 2 × frozen stock solution into each cell/ml, blowing, and packaging to obtain 0.3-1 × 10 cell 6 And (4) carrying out one cell per cell, carrying out overnight at-80 ℃ in a freezing storage box, and transferring to a liquid nitrogen tank for long-term freezing storage the next day.
More preferably, the Muller cell culture medium is a DMEM basal medium added with 10% fetal bovine serum by volume fraction, 1% GlutaMAX by volume fraction, 1% nonessential amino acids by volume fraction and 1% antibacterial fungicide by volume fraction.
More preferably, the 2 Xfreezing medium in step d is prepared by mixing fetal bovine serum, Muller cell culture medium and dimethyl sulfoxide in a volume ratio of 3:1: 1.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention explores the ability of retinal organoids at different developmental stages to separate and expand human muller cells, and finds that retinal organoids induced to differentiate to a late stage, such as after day 120, are preferred muller cell donors;
(2) by utilizing the method, the human Muller cells are obtained through differentiation of the human pluripotent stem cells, the species is humanized, the result error caused by species difference in subsequent research is reduced, and the human pluripotent stem cells have the capability of unlimited proliferation, so that the preparation method can continuously obtain the Muller cells and make up the limitation of donor shortage of the Muller cells separated from human tissues;
(3) the human Muller cells obtained by the method have no pollution of other retina cells, astrocytes and other non-Muller cells through identification, and have high purity;
(4) with the present invention, obtaining human muller cells by this method reduces the risk of immune rejection and disease transmission, e.g. using patient-specific human induced pluripotent stem cells, i.e. patient-specific muller cells can be obtained by differentiation by the method of the present invention;
(5) the obtained human Muller cells have better passage amplification capacity, can be continuously amplified for more than 10 generations, can produce a large number of cells in a short period, and can be stored by a simple cryopreservation method for future batch production and application;
(6) by using the invention, the obtained human Muller cells still keep stable phenotype after multiple passages;
(7) the present invention can provide useful cell tool or seed cell for the physiological and pathological state characteristics and functions of Muller cell, the mechanism of retina disease, cell treatment research, etc.
Description of the drawings:
FIG. 1 is a schematic representation of the preparation of Muller cells from human pluripotent stem cell-derived retinal organoids.
FIG. 2 is a typical photomorphogram of the process of differentiation of human pluripotent stem cells into three-dimensional retinal organoids. Wherein (A) is a human induced pluripotent stem cell; (B) is an embryoid body; (C) is a three-dimensional retinal organoid. Wherein, RPE: retinal pigment epithelial cells; NR: the neural retina layer. Scale bar 100 μm.
Fig. 3 shows a neural retina patch (a) obtained by mechanical separation from a three-dimensional retina organoid, and a single retinal cell (B) obtained after digestion of the neural retina patch. Scale bar 200 μm.
FIG. 4 is a bright field diagram of adherent expansion culture of retinal cells isolated from retinal organoids at different stages of development. Scale bar 200 μm.
FIG. 5 is an identification of immunofluorescence staining of retinal cells obtained from retinal organoid expansion at different stages of development. Scale bar 50 μm.
FIG. 6 is a graph showing the doubling time of serial passage expansion cultures of retinal cells or Muller cells obtained from expansion in retinal organoids at different stages of development.
FIG. 7 is a diagram showing the identification of RNA-seq of human Muller cells prepared by the method of the present invention. Wherein (A) is a PCA diagram; (B) a volcano plot; (C-H) is a heat map, and (D-H) is a heat map of the expression of the marker genes associated with Photoreceptor Cells (PC), Amacrine Cells (AC)/Horizontal Cells (HC), ganglion cells (RGC), Bipolar Cells (BC), and Muller cells (MGC), respectively. iMGC-1, -2, -3 are three replicate samples of human Muller cells prepared by the method of the invention; RO-1, -2, -3 are three replicate samples of neural retinal membranes isolated from three-dimensional retinal organoids.
FIG. 8 shows the expression of astrocyte markers in human muller cells derived from human tissue (MIO-M1) and obtained by the method of the Invention (iMGC). Scale bar 50 μm.
FIG. 9 shows the cell characterization of human Muller cells obtained by the method of the present invention after subculture expansion. Wherein (A) is an immunofluorescence staining pattern of Muller cell markers SOX9 and GS in different generations of human Muller cells prepared by the method; (B) different generations of human Muller cell RNA-seq analysis samples correlation heatmaps obtained for the methods of the invention. P1-1, -2, -3 are three replicate samples of the 1 st generation of human Muller cells prepared by the method of the invention; p5-1, -2, -3 are three replicate samples of 5 th generation human Muller cells obtained by the method of the invention.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: method for preparing Muller cells derived from human pluripotent stem cells
A schematic of the preparation and expansion passages of human pluripotent stem cell-derived Muller cells is shown in FIG. 1.
1. Maintenance culture of human induced pluripotent stem cells (hipscs)
Cell: BC1 hiPSC and BC1-GFP hiPSC, which were gifted by Chilobrachys professor (university of science and technology of China).
Reagents and consumables:
1) mTeSR1 medium: STEM CELL, # 05851;
2)EDTA：Invitrogen，15575-038；
3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;
4)Matrigel：Corning，354277；
5) a six-hole plate: FALCON, 353046;
6) centrifuging the tube: FALCON, 352096.
The instrument comprises the following steps:
1)CO 2 an incubator: SANYO, MCO-20A 1C;
2) and (3) inverting the microscope: nikon, TS 100.
The method comprises the following specific steps:
BC1 hiPSCs and BC1-GFP hiPSCs are maintained and cultured in mTeSR1 medium, and passage is carried out when the fusion degree is about 80% -90%, and the specific operation is as follows: mTeSR1 medium was aspirated, washed with sterile PBS, and digested for 3-7min at 37 deg.C with the addition of 0.5mM EDTA which had been rewarmed to 37 deg.C. EDTA was aspirated, cells were blown down with a small amount of mTeSR1 medium, and plated onto Matrigel-coated plates at 37 ℃ with 5% CO 2 And (5) culturing in an incubator.
And (4) analyzing results:
human induced pluripotent stem cells were cultured adherent to the wall, growing in typical flat clones with clear borders and dense cells within the clones (fig. 2A).
2. Formation of embryoid bodies and induction differentiation to obtain three-dimensional retina organs
Reagents and consumables:
1) mTeSR1 medium: STEM CELL, # 05851;
2)Matrigel：Corning，354277；
3) DMEM/F12 basal medium: gibco, C11330500 BT;
4) DMEM basal medium: gibco, C11995500 BT;
5) non-essential amino acids: gibco, 11140-050;
6)GlutaMAX：Gibco，35050-061；
7) b27 (without vitamin a): gibco, 17504044;
8) heparin: sigma, 2mg/mL in PBS;
9) taurine: sigma, # T-0625;
10) fetal bovine serum: natocor, 10099-141;
11) antibacterial and antifungal agents: gibco, 15240;
12) culture dish: BIOFIL, TCD 000100; falcon, 353003
13)1ml syringe: shanghai Kangdelai Enterprise development group GmbH, batch K20191207;
14) n2 additive: gibco, 17502048.
15)(-)-Blebbistatin：Sigma，B0560；
The instrument comprises the following steps:
1)CO 2 an incubator: SANYO, MCO-20A 1C;
2) and (3) inverting the microscope: nikon, TS 100.
The construction and induced differentiation of embryoid bodies to obtain three-dimensional retinal organoids was carried out by reference to published methods (Zhong et al, 2014; Li et al, 2018; Guan et al, 2021).
The method comprises the following specific steps:
when the fusion degree of BC1-GFP hipSCs is 80% -90%, the mTeSR1 medium is aspirated, washed with PBS, and then 0.5mM EDTA which has been rewarming to 37 ℃ is added, 5% CO is added at 37 ℃ 2 Digesting for 3-7 min. Absorbing EDTA, blowing down cells with mTeSR1 culture medium containing 10 μ M (-) -Blebbistatin to obtain small cell mass, transferring the cell mass into low adsorption culture dish, adding mTeSR1 culture medium containing 10 μ M (-) -Blebbistatin, shaking up, 37 deg.C, 5% CO 2 Culturing and constructing embryoid bodies.
The day of construction of the embryoid bodies was taken as day 0 of induced differentiation of the three-dimensional retinal organoids. The next day is marked as day 1, and so on.
On days 1-3, the total volume of mTeSR1 medium was gradually changed to neural induction medium (ingredient: DMEM/F12 basal medium supplemented with 1% by volume of N2 additive, 1% by volume of non-essential amino groups, heparin to a final concentration of 2. mu.g/mL). The method comprises the following specific steps: inoculating the embryoid bodies to mTeSR1 culture medium containing 10 μ M (-) -Blebbistatin on day 0 for suspension culture; on the first day, medium mTeSR1 containing 10. mu.M (-) -Blebbistatin was replaced with medium A (neural induction medium mixed with mTeSR1 at a volume ratio of 1: 3); the next day, medium a was replaced with medium B (nerve induction medium mixed with mTeSR1 at a volume ratio of 1: 1); on the third day, medium B was replaced with neural induction medium. The embryoid bodies were inoculated on days 5 to 7 onto Matrigel-coated plates for further culture.
The medium was changed to a retinal differentiation medium on day 16 (composition: DMEM/F12 basal medium supplemented with 35% by volume DMEM basal medium, 2% by volume B27 supplement (containing no vitamin a), 1% by volume antibacterial antifungal agent, 1% by volume non-essential amino acids).
On day 28, the retinal tissue with good structure can protrude out of the bottom surface of the culture dish, the retinal tissue is picked up by a 1ml syringe and is cultured in suspension in a low adsorption culture dish containing a retinal culture medium 1 (the components are: DMEM/F12 basal medium, DMEM basal medium with the volume fraction of 40%, B27 additive (without vitamin A) with the volume fraction of 2%, antibacterial fungicide with the volume fraction of 1%, non-essential amino acid with the volume fraction of 1%, fetal calf serum with the volume fraction of 10%, GlutaMAX with the volume fraction of 1% and taurine with the final concentration of 100 MuM) to obtain the three-dimensional retinal organs. The step of picking up the retinal tissue specifically comprises the following steps: finding a target retinal tissue under a microscope, generally in a horseshoe-shaped transparent bulge, holding a 1ml syringe by a hand, moving a syringe needle to the position of the retinal tissue under the microscope, drawing a circle around the target retinal tissue by using a needle point, gently picking up a cell layer around the retinal tissue from one side, separating the retinal tissue from a dish bottom and completely suspending in a retinal culture medium 1 under the condition of not damaging the original structural form of the retinal tissue, and changing the liquid every other day.
On day 90, the medium was changed to retina medium 2 (DMEM/F12 basal medium supplemented with 1% by volume of N2 supplement, 1% by volume of antibacterial antifungal agent, 1% by volume of nonessential amino acids, 10% by volume of fetal bovine serum, 1% by volume of GlutaMAX, and 100. mu.M final concentration of taurine), and the medium was cultured in suspension for a long period of time, with the medium changed every other day.
And (4) analyzing results:
the small cell mass formed by the digestion of human induced pluripotent stem cells was cultured in suspension to form a clear spheroid embryoid body (FIG. 2B). After the embryoid bodies are differentiated, three-dimensional retina organoids (fig. 2C) at different developmental stages, which include retinal pigment epithelial cells and neural retina layers, can be obtained according to the number of induced differentiation days. The three-dimensional retina organoids can be cultured in vitro for a long time, and the longest observation period is more than 1 year.
3. Separating neural retina sheets from three-dimensional retina organs, and digesting to obtain single retinal cell suspension
Reagents and consumables:
1) DMEM/F12 basal medium: gibco, C11330500 BT;
2)N2：Gibco，17502048；
3) non-essential amino acids: gibco, 11140-050;
4)GlutaMAX：Gibco，35050-061；
5) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;
6) taurine: sigma, # T-0625;
7) fetal bovine serum: natocor, 10099-141;
8) antibacterial and antifungal agents: gibco, 15240;
9) culture dish: BIOFIL, TCD 000100;
10)1ml syringe: shanghai Kangdelai Enterprise development group GmbH, batch K20191207;
11) the papain kit comprises: Worthington-Biochem; LK 003153;
12) DMEM basal medium: gibco, C11995500 BT;
the instrument comprises the following steps:
1) and (3) inverting the microscope: nikon, TS 100.
The method comprises the following specific steps:
three-dimensional retina organoids of different developmental stages (D60, D90, D120, D150) were placed in a low adsorption petri dish containing a retina medium 1 or a retina medium 2, other non-neural retina organoid tissues and retinal pigment epithelial cells adhered to the retina organoids were removed under a microscope with a 1ml syringe, then mechanical separation was performed along the junction between the neural retina layer and the non-neural retina layer, the non-neural retina layer was discarded with a rubber head dropper, and the neural retina sheet was obtained and cut into fragments of uniform size. The pieces were washed 3 times with 5ml sterile PBS and the supernatant removed. Digesting for 30-90min in water bath at 37 ℃ according to the instruction of a papain kit. The single cell suspension was obtained by pipetting with a 10ml pipette, centrifuging at 300g for 10min and removing the supernatant. Stop solution was added to stop digestion. Centrifuge at 300g for 10min at room temperature and remove the supernatant. The cells were resuspended in Muller cell culture medium (composition: DMEM basal medium supplemented with 10% by volume fetal bovine serum, 1% by volume GlutaMAX, 1% by volume non-essential amino acids and 1% by volume antibacterial fungal agent) to obtain retinal cell suspension.
And (4) analyzing results:
by mechanical separation, uniformly sized, clear neural retinal patches were obtained from three-dimensional retinal organoids (fig. 3A), and well-activated single retinal cells were obtained by digestion (fig. 3B).
4. Amplification culture and immunofluorescence identification of human muller cells derived from human pluripotent stem cells
(1) Amplification culture:
reagents and consumables:
1) DMEM basal medium: gibco, C11995500 BT;
2) non-essential amino acids: gibco, 11140-050;
3)GlutaMAX：Gibco，35050-061；
4) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;
5) antibacterial and antifungal agents: gibco, 15240;
6) fetal bovine serum: natocor, 10099-141;
7)Matrigel：Corning，354277；
8) a six-hole plate: FALCON, 353046;
9) centrifuging the tube: FALCON, 352096.
The instrument comprises the following steps:
1)CO 2 an incubator: SANYO, MCO-20A 1C;
2) and (3) inverting the microscope: nikon, TS 100.
The method comprises the following specific steps:
the retinal cell suspension obtained by digesting neural retina membranes separated from retina organoids at different developmental stages (D60, D90, D120, D150) is used for>1×10 4 Individual cell/cm 2 The cells were cultured in a matrix-coated 6-well cell culture plate containing Muller cell culture medium (composition: DMEM basal medium supplemented with 10% by volume fetal bovine serum, 1% by volume GlutaMAX, 1% by volume nonessential amino acids and 1% by volume of an antibacterial agent) for amplification, and the growth characteristics of the cells were recorded by an optical microscope.
And (4) analyzing results:
the neural retina sheets separated from the retina organoids in different development stages are digested to obtain retina cells, and the retina cells are inoculated in a culture plate for adherent culture and amplification. Retinal cells from early (D60, D90) and late (D120, D150) three-dimensional retinal organoids can both grow and expand adherently in muller cell culture medium, but exhibit different growth patterns. As shown in fig. 4, cells from early (D90) retina organoids, cultured adherent in muller cell culture medium, were observed to be more viable, rapidly proliferating and reaching about 90% confluence within one week on day 1; whereas cells from late (D150) retinal organoids, only a few cells were observed to survive on day 1, growing as single clones, reaching about 90% confluence up to 2 weeks.
(2) And (3) immunofluorescence identification:
reagents and consumables:
1) PBS powder: boster immunolader, AR 0030;
2) PFA powder: sigma, P6148-1 KG;
3) DAPI dye: NY809, 1:1000, Dong ren chemical technology (Shanghai) Co., Ltd;
4)TritonX-100：MP Biomedicals,LLC；194854；
5)Donkey serum：Hyclone，XT-100；
6) rabbit anti-SOX 9 primary antibody (1:250, A19710, ABClonal, China); murine anti-GS primary antibody (1:200, 610517, BD, usa); murine anti-CRX primary antibody (1:800, H00001406-M02, Abnova, China); sheep resistance to VSX2 primary (1: 200; ab9016, Millipore, USA); murine anti-PAX 6 primary antibody (1:50, 3B5, chinese DSHB); rabbit anti-vimentin primary antibody (1:50, BM4029, doctor, China); rabbit anti-nestin primary antibody (1:200, ab82375, Abcam, UK);
7) donkey anti-rabbit Alexa Fluor-555 labeled secondary antibody (1:500, A31572, U.S. Thermo Fisher Scientific), donkey anti-mouse Alexa Fluor-555 labeled secondary antibody (1:500, A31570, U.S. Thermo Fisher Scientific); donkey anti-sheep Alexa Fluor-555 labeled secondary antibody (1:500, A21436, U.S. Thermo Fisher Scientific)
8) Anti-fluorescence quenching encapsulated tablet: biyunyan, P0128M-2;
9)coverslip：ThermoFisher scientific，12-545-80。
the instrument comprises the following steps:
1) inverted fluorescence microscopy: ZEISS, HAL 100;
2) a slide scanner: ZEISS, Axio Scan.21.
The method comprises the following specific steps:
retinal cells from different developmental stages (D60, D90, D120, D150) of the retinal organoids were seeded on Matrigel-coated coverslip according to the expansion culture method of this example. Retinal cells seeded on coverslip were fixed with 4% PFA and immunofluorescence detected. The immunofluorescence detection method comprises the following steps: sealing and permeating 0.25% TritonX-100 containing 10% donkey serum at room temperature for 1h, dripping corresponding primary antibody, incubating overnight at 4 ℃, washing out the primary antibody, dripping corresponding secondary antibody, and incubating at room temperature in a dark place for 1 h. Washing the secondary antibody, adding a DAPI solution, dyeing the nucleus for 5min in a dark place at room temperature, washing the DAPI, sealing the chip by an anti-fluorescence quenching sealing agent, and observing and recording the fluorescence expression condition of the corresponding channel under a fluorescence microscope.
And (4) analyzing results:
immunofluorescent staining results showed that there was a difference in expression of cell markers between cells obtained after adherent expansion of cells from early (D60, D90) and late (D120, D150) retinal organoids in muller cell culture medium. After adherent expansion culture of cells from early (D90) retinal organoids in muller cell culture medium, some cells expressed muller cell markers SOX9, vimentin and nestin, but not the key mature muller cell marker GS, and some expressed retinal precursor cells and the bipolar cell marker VSX2, retinal precursor cells, ganglion cells, horizontal cells and the amacrine cell marker PAX6 and the photoreceptor cell marker CRX (fig. 5). These results show that retinal cells from early retinal organoids, after adherent expansion culture in Muller cell culture medium, give a heterogeneous cell mixture with low purity. While retinal cells from late (D150) retinal organoids, after adherent expansion culture in Muller cell culture medium, mainly expressed the Muller cell markers SOX9, vimentin and nestin, in particular expressed their maturation markers GS, but hardly expressed other retinal cell markers such as VSX2, PAX6 or CRX (FIG. 5), indicating that retinal cells from late retinal organoids, after adherent expansion culture in Muller cell culture medium, obtained Muller cells of high purity.
5. Subculturing and cryopreserving retinal cells or human muller cells from retinal organoids at different developmental stages
Reagents and consumables:
1) DMEM basal medium: gibco, C11995500 BT;
2) non-essential amino acids: gibco, 11140-050;
3)GlutaMAX：Gibco，35050-061；
4) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;
5) antibacterial and antifungal agents: gibco, 15240;
6) fetal bovine serum: natocor, 10099-141;
7)Matrigel：Corning，354277；
8) a six-hole plate: FALCON, 353046;
9) centrifuging the tube: FALCON, 352096;
10)TRYPSIN 0.25％EDTA：Gibco，25200056；
11) dimethyl sulfoxide: sigma; d2650-100 ML;
12) cell cryopreservation tube: thermo fisher scientific; 375418, respectively;
the instrument comprises the following steps:
1)CO 2 an incubator: SANYO, MCO-20A 1C;
2) and (3) inverting the microscope: nikon, TS 100;
3) -80 ℃ refrigerator: thermo Scientific; 88000, respectively;
4) a low-speed centrifuge: flying pigeon brand; TDL-40B.
The method comprises the following specific steps:
passage: according to the preparation method of this example, retinal cells obtained by segregating and expanding retinal organoids at different developmental stages were serially passaged and cultured until about 90% confluence was reached. The culture supernatant was removed and washed 3 times with 1 ml/well of 1 XPBS. Adding TRYPSIN 0.25% EDTA at 1 ml/well, and digesting at 37 deg.C for 5-10 min. The cell suspension was obtained by gentle blowing with a 1ml pipette, digestion was stopped by adding 10ml of Muller cells, centrifugation was carried out at 300g for 10min, and the supernatant was removed. After adding 1ml of Muller cells for resuspension, in>1×10 4 Individual cell/cm 2 Seeded on Matrigel-coated 6-well cell culture plates containing muller cell culture medium.
Freezing and storing: the resulting Muller cell suspension from the digestion is adjusted to a concentration of 0.5-1X 10 according to the counting results 6 Dropping equal volume of 2 × freezing medium (containing fetal calf serum, Muller cell culture medium and dimethyl sulfoxide at a volume ratio of 3:1:1) while shaking per cell/ml, blowing, and packaging to give 0.3-1 × 10 6 Individual cell/branch. And (4) in a freezing storage box, standing overnight at-80 ℃, and transferring to a liquid nitrogen tank for long-term freezing storage the next day.
And (4) analyzing results:
the cell doubling time curves show that retinal cells from different developmental stages of the retinal organoids were expanded in Muller cell culture medium and exhibited different growth characteristics. Retinal cells from early-developing (D60, D90) retinal organoids, although the early-generation cells proliferate rapidly, lose their proliferative activity rapidly and have poor passability, up to 6 generations; retinal cells from the retinal organoids after the late stage (D120, D150) showed selective clonal growth with better passage expansion capacity for more than 10 passages (fig. 6).
In combination with the aforementioned immunofluorescence assay results (FIG. 5), retinal organoids after induced differentiation for 120 days are the preferred donors of Muller cells.
RNA-seq analysis of the transcriptome characteristics of human Muller cells obtained by the method of the invention
Reagents and consumables:
1) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;
2)TRYPSIN 0.25％EDTA：Gibco；25200056；
3) trizol solution: sigma; t9424-200 ML.
The instrument comprises the following steps:
1) -80 ℃ refrigerator: thermo Scientific; 88000, respectively;
2) a low-speed centrifuge: flying pigeon brand; TDL-40B.
The method comprises the following specific steps:
human Muller cells prepared by the method of the present invention were cultured adherent to the cells, and when about 90% confluence was reached, the culture medium supernatant was removed and washed 3 times with 1 ml/well of 1 XPBS. Adding TRYPSIN 0.25% EDTA at 1 ml/well, and digesting at 37 deg.C for 5-10 min. The cell suspension was obtained by gentle blowing with a 1ml pipette, digestion was stopped by adding 10ml of Muller cell culture medium, centrifugation was carried out at 300g for 10min, and the supernatant was removed. Washing with 1ml of 1 XPBS for 3 times, centrifuging to remove PBS to obtain the human Muller cell sediment prepared by the method of the invention, quickly freezing with liquid nitrogen, and transferring to a refrigerator at-80 ℃ for storage. Similarly, according to the method for separating the neural retina membranes of the present example, after the neural retina membranes are obtained by separation, 1ml of 1 XPBS is added for washing 3 times, and after the PBS is removed by centrifugation, the neural retina membrane precipitate is obtained, is frozen by liquid nitrogen, and is stored in a refrigerator at-80 ℃. The collected human muller cells and neural retina plate samples were sent to guangzhou deo biotechnology ltd for RNA sequencing and result analysis.
And (4) analyzing results:
as can be seen from FIG. 7, the human Muller cells (iMGCs) prepared by the method of the present invention have a larger gene expression difference with neural Retina Sheets (ROs) derived from late retinal organoids, and compared with the neural retina sheets, the expression of Muller cell-associated marker genes in the human Muller cells prepared by the method of the present invention is significantly up-regulated, while the expression of associated marker genes of other retinal cells is significantly down-regulated, further illustrating that the human Muller cells obtained by the method of the present invention are cultured and passaged in Muller cell culture medium adherent to reduce the contamination of other retinal cells, and the human Muller cells with high purity are obtained.
7. Immunofluorescence analysis of contamination of astrocytes in human Muller cells prepared according to the method of the invention
Cell: human retinal tissue-derived MIO-M1 cell line, purchased from North Nam Biotech;
reagents and consumables:
1) PBS powder: boster immunolader, AR 0030;
2) PFA powder: sigma, P6148-1 KG;
3) DAPI dye: NY809, 1:1000, Dong ren chemical technology (Shanghai) Co., Ltd;
4)TritonX-100：MP Biomedicals,LLC；194854；
5)Donkey serum：Hyclone，XT-100；
6) rabbit anti-GFAP primary antibody (1:50, BM4287, bosch de china);
7) donkey anti-rabbit Alexa Fluor-555 labeled secondary antibody (1:500, A31572, Thermo Fisher Scientific, USA);
8) anti-fluorescence quenching encapsulated tablet: biyunyan, P0128M-2;
9)coverslip：ThermoFisher scientific，12-545-80；
10)1640 basic medium: gibco, C11875500 BT;
11) fetal bovine serum: natocor, 10099-141;
12) antibacterial and antifungal agents: gibco, 15240.
The instrument comprises the following steps:
1) inverted fluorescence microscopy: ZEISS, HAL 100;
2) a slide scanner: ZEISS, Axio Scan.21.
The method comprises the following specific steps:
human Muller cells prepared according to the method of the present invention were seeded on Matrigel-coated coverslip according to the amplification culture method of this example. MIO-M1 at 1X 10 4 Individual cell/cm 2 The culture medium is inoculated in a medium containing MIO-M1 (component: 1640 basic medium added with 10% fetal calf serum and 1% antibacterial antifungal agent)) Is coated on coverslip.
Human Muller cells and MIO-M1 cells prepared by the method of the present invention inoculated on coverslip were fixed with 4% PFA and then subjected to immunofluorescence detection. The immunofluorescence detection method comprises the following steps: sealing and permeating 0.25% TritonX-100 containing 10% donkey serum at room temperature for 1h, dripping corresponding primary antibody, incubating overnight at 4 ℃, washing out the primary antibody, dripping corresponding secondary antibody, and incubating at room temperature in a dark place for 1 h. Washing the secondary antibody, adding a DAPI solution, dyeing the nucleus for 5min in a dark place at room temperature, washing the DAPI, sealing the chip by an anti-fluorescence quenching sealing agent, and observing and recording the fluorescence expression condition of the corresponding channel under a fluorescence microscope.
And (4) analyzing results:
the immunofluorescence staining results (FIG. 8) show that when compared with the Muller cell line MIO-M1 derived from human tissue, the human Muller cells obtained by the method of the present invention do not express the astrocyte marker GFAP, while the MIO-M1 cells still express a small amount of GFAP, further illustrating that the human Muller cells with high purity can be obtained by the method of the present invention, and the problem that the Muller cells separated and amplified from human tissue are easy to mix with other cell components is solved.
8. Immunofluorescence and RNA-seq analysis identification of phenotypic stability of human Muller cells prepared by the method of the invention after passage
The method comprises the following specific operations:
the phenotypic stability of human Muller cells obtained by the method of the present invention after passaging was characterized by reference to the immunofluorescence and RNA-seq methods described in this example.
And (4) analyzing results:
through immunofluorescence identification, the human Muller cells of different generations prepared by the method of the invention all abundantly express classical markers of Muller cells such as SOX9 and GS (FIG. 9A). And RNA-seq analysis shows that the transcriptome of human Muller cells of different generations obtained by the method has no significant difference (FIG. 9B), which confirms that the human Muller cells obtained by the method still maintain stable phenotype after multiple passages.
Claims (10)
1. A method for producing Muller cells derived from human pluripotent stem cells, comprising the steps of:
a. inducing and differentiating the human pluripotent stem cells to obtain three-dimensional retina organs;
b. removing non-retina organoid tissues, retinal pigment epithelial cells and non-neural retina layers from the three-dimensional retina organoids obtained in the step a to obtain neural retina sheets;
c. b, digesting the neural retina sheet obtained in the step b to obtain a retina single cell suspension;
d. and d, performing adherent culture on the retinal single cell suspension obtained in the step c, removing non-Muller cells to obtain Muller cells induced by the human pluripotent stem cells, and performing passage expansion and cryopreservation on the Muller cells.
2. The method of claim 1, wherein the three-dimensional retinal organoid is a human pluripotent stem cell-derived three-dimensional retinal organoid induced to differentiate to an advanced stage.
3. The method according to claim 1, wherein in step a, the induced differentiation of human pluripotent stem cells to obtain three-dimensional retinal organoids is performed by digesting human pluripotent stem cells into small cell clusters, placing the small cell clusters in mTeSR1 medium containing 10 μ M (-) -Blebbistatin to form embryoid bodies, gradually replacing the medium with a neural induction medium, and then inoculating the embryoid bodies into a Matrigel-coated culture dish for adherent culture; differentiating to 16 days, culturing with a retinal differentiation culture medium until retinal tissues protruding out of the bottom surface of the culture dish are formed, picking up the retinal tissues, inoculating the retinal tissues into the retinal culture medium 1, and performing suspension culture to obtain the three-dimensional retinal organoids derived from the human pluripotent stem cells; starting from the 90 th day of differentiation, the culture medium was changed to retina culture medium 2, and suspension culture was continued for a long period;
the nerve induction culture medium is heparin, wherein 1% of N2 additive in volume fraction, 1% of non-essential amino acid in volume fraction and 2 mug/mL are added into a DMEM/F12 basal culture medium;
the retina differentiation medium is a DMEM basic medium with 35% volume fraction, a B27 additive without vitamin A with 2% volume fraction, an antibacterial fungicide with 1% volume fraction and nonessential amino acid with 1% volume fraction added in a DMEM/F12 basic medium;
the retina culture medium 1 is prepared by adding 40% volume fraction DMEM basal medium, 2% volume fraction B27 additive without vitamin A, 1% volume fraction antibacterial fungicide, 1% volume fraction nonessential amino acid, 10% volume fraction fetal bovine serum, 1% volume fraction GlutaMAX and 100 mu M final concentration taurine into DMEM/F12 basal medium;
the retina culture medium 2 is prepared by adding 1% of N2 additive, 1% of antibacterial fungicide, 1% of nonessential amino acid, 10% of fetal calf serum, 1% of GlutaMAX and 100 mu M of taurine into a DMEM/F12 basal culture medium.
4. The method according to claim 1, wherein in the step b, the non-retinal organoid tissue, retinal pigment epithelial cells and non-neural retinal layer of the three-dimensional retinal organoid obtained in the step a are removed to obtain the neural retinal membrane, the three-dimensional retinal organoid obtained by differentiation in the step a is placed in a low adsorption culture dish containing a retinal culture medium 2, and the non-retinal organoid tissue and retinal pigment epithelial cells are mechanically separated and discarded; and then mechanically separating along the junction between the neural retina layer and the non-neural retina layer, and discarding the non-neural retina layer to obtain the neural retina membrane.
5. The method according to claim 1, wherein in the step c, the neural retina fragments obtained in the step b are digested by papain to obtain the retinal single cell suspension.
6. The method according to claim 1, wherein in step d, the retinal single cell suspension obtained in step c is cultured adherently, and non-Muller cells are removed to obtain Muller cells induced by human pluripotent stem cells, and the obtained retinal single cell suspension is inoculated on a Matrigel-coated cell culture plate containing Muller cell culture medium; carrying out amplification culture on the surviving cells to obtain Muller cells;
the Muller cell culture medium is prepared by adding 10% fetal calf serum by volume fraction into a DMEM basal medium, 1% GlutaMAX by volume fraction, 1% nonessential amino acid by volume fraction and 1% antibacterial fungicide by volume fraction.
7. The preparation method according to claim 3, wherein the human pluripotent stem cells are digested into small cell clusters, the small cell clusters are placed in mTeSR1 culture medium containing 10 μ M (-) -Blebbistatin to form embryoid bodies, the human pluripotent stem cells are cultured in mTeSR1 culture medium in an adherent manner, when the clone grows to occupy 80% -90% of the area of the bottom of a hole, the differentiated cells are scraped, the mTeSR1 culture medium is aspirated, after being rinsed with sterile PBS and digested with 0.5mM EDTA solution, the EDTA is aspirated, the mTeSR1 culture medium containing 10 μ M (-) -Blebbistatin is taken out and blown down to obtain small cell clusters, and the small cell clusters are placed in mTeSR1 culture medium containing 10 μ M (-) -Blebbistatin to form embryoid bodies in a suspended culture.
8. The preparation method according to claim 3, wherein the gradual replacement with the neural induction medium is carried out by recording the day of establishing the embryoid body as the day of induced differentiation, recording the next day as the day of induced differentiation, and so on; the embryoid bodies are inoculated to mTeSR1 culture medium containing 10 mu M (-) -Blebbistatin for suspension culture on day 0, the mTeSR1 culture medium containing 10 mu M (-) -Blebbistatin is replaced by a culture medium A on day 1, the culture medium A is prepared by mixing a nerve induction culture medium and mTeSR1 according to the volume ratio of 1:3, the culture medium A is replaced by a culture medium B on day 2, the culture medium B is prepared by mixing the nerve induction culture medium and mTeSR1 according to the volume ratio of 1:1, and the culture medium B is replaced by the nerve induction culture medium on day 3.
9. The method according to claim 3, wherein the retinal tissue is picked up by drawing a circle around the target retinal tissue, gently picking up the cell layer around the retinal tissue from one side, and completely suspending the retinal tissue in the retinal culture medium 1 without damaging the original structural form of the retinal tissue.
10. The method according to claim 1, wherein in step d, the passage expansion is performed by digesting Muller cells with fusion degree of 80-90%, and then inoculating the digested Muller cells on a Matrigel-coated cell culture plate containing Muller cell culture medium for culture;
the cryopreservation is to drop equal volume of cryopreservation liquid into Muller cell suspension obtained by digestion; the cryopreservation solution is 2 Xcryopreservation solution and is obtained by mixing fetal bovine serum, Muller cell culture medium and dimethyl sulfoxide in a volume ratio of 3:1: 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116121173A (en) * | 2023-03-14 | 2023-05-16 | 广州湾区生物基因科技有限公司 | Eye tissue organoid and derived cell line thereof, preparation method and application thereof |
| CN116121173B (en) * | 2023-03-14 | 2023-09-01 | 广州湾区生物基因科技有限公司 | Eye tissue organoid and derived cell line thereof, preparation method and application thereof |
| CN117286106A (en) * | 2023-09-14 | 2023-12-26 | 广州湾区生物基因科技有限公司 | Mouse retina organoid, terminally differentiated cell line, construction method and application thereof |
| CN117286106B (en) * | 2023-09-14 | 2024-05-24 | 广州湾区生物基因科技有限公司 | Construction method of mouse retina organoids |
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