WO2016129654A1 - Method for preparing corneal endothelial precursor cells - Google Patents

Abstract

　The purpose of the present invention is to provide: a method for preparing corneal endothelial cells having a pump function and precursor cells thereof; corneal endothelial cells and precursor cells thereof prepared using said method; and a method for producing a corneal endothelial cell sheet for transplant, using said corneal endothelial cells and the precursor cells thereof. By culturing corneal endothelial cells under low serum conditions, it becomes possible to grow corneal endothelial precursor cells while maintaining productivity and undifferentiated properties, and the aforementioned problem was resolved by using the corneal endothelial cells in an unchanged state or by inducing differentiation in same.

Description

Method for preparing corneal endothelial progenitor cells

The present invention relates to a method for preparing corneal endothelial progenitor cells. More specifically, the present invention relates to a method for preparing corneal endothelial progenitor cells, comprising culturing corneal endothelial cells in a low-serum culture medium. The present invention also relates to a corneal endothelial progenitor cell prepared by the above preparation method and a method for producing a corneal endothelial cell material in which the corneal endothelial progenitor cell is supported on a carrier.

The cornea is a layered tissue that constitutes the eye, and is composed of five layers with the corneal epithelium as the outermost layer and the corneal endothelium as the innermost layer. Corneal epithelial cells are known to have high proliferation ability and high regenerative power, while corneal endothelial cells have poor regenerative power. The corneal endothelium has a pump function of actively transporting water from the corneal stroma to the anterior chamber, thereby preventing white turbidity (corneal turbidity) caused by water permeating into the cornea. When the corneal endothelium is damaged due to various factors and the number of corneal endothelial cells decreases, the corneal endothelial cells cannot compensate for the decreased location by cell proliferation, increase the cell surface area by extension movement and expansion, It tries to make up for the damaged part. As a result, when the density per unit area of the corneal endothelial cells decreases and falls below a certain density, a sufficient pump function cannot be maintained, resulting in corneal opacity and bullous keratopathy.

Bullous keratosis is a serious disease that leads to blindness, and treatment requires corneal transplantation. However, donor shortage is a chronic problem and often causes rejection after surgery. In transplantation treatment, the transparent cure rate is not high, and there are problems such as insufficient corrective visual acuity after the operation. Therefore, treatment by corneal transplantation is not a perfect treatment method.

On the other hand, with the advancement of regenerative medicine, research for producing cultured corneal endothelial cell sheets for transplantation has also been conducted. Patent Document 1 (Japanese Patent Laid-Open No. 2005-229869) relates to a method for reconstructing a cornea in which isolated and cultured corneal endothelial cells are cultured and transplanted. Patent Document 2 (WO2011 / 096593) has found that an ascorbic acid derivative improves the growth rate in the culture of corneal endothelial cells. Non-patent document 1 (STEM CELLS AND DEVELOPMENT) has isolated human corneal endothelial progenitor cells based on the expression of p75 neurotrophin receptor (also referred to as p75NTR or P75), and thus isolated human corneal endothelium. It is disclosed that progenitor cells have sufficient proliferation and can be used for the production of transparent corneal endothelial cell sheets. Patent Document 3 (WO2013 / 012087) describes that by using a specific serum-free culture solution, p75-positive corneal endothelial precursor details with high proliferation ability and an undifferentiated state can be obtained. In Patent Document 4 (WO2014 / 007402) and Non-patent Document 3 (STEM CELLS 2013; 31: 1396-1407), the expression of LGR5 maintains the endothelial cell phenotype, and the transition of endothelial mesenchyme via Wnt signal Furthermore, it has been disclosed that R-spodins, which are ligands of LGR5, contribute to suppression of differentiation and / or promotion of proliferation of corneal endothelial cells. Thus, a method for proliferating corneal endothelial cells has been studied. However, corneal endothelial cells have extremely limited proliferative properties, and depending on the culture conditions, different cells called endometrial mesenchymal transition (EndMT). It is known that culturing is very difficult because the transformation into, and the pump function is lost. Even when the methods of Non-Patent Documents 1 and 3 and Patent Documents 2 to 4 were used, the proliferation ability was not sufficient, and the cobblestone-like corneal endothelial cells or their progenitor cells could not be grown. Therefore, a method for preparing corneal endothelial cells having a pump function by sufficiently growing them is still desired.

JP 2005-229869 A International Publication No. 2011/096593 International Publication No. 2013/012087 International Publication No. 2014/007402

STEM CELLS AND DEVELOPMENT 2014; 23: 2190-2201, The Journal of Biological Chemistry, (2004) vol.279, No.31, pp.32325-32332 STEM CELLS 2013; 31: 1396-1407

In view of the above circumstances, the problem to be solved by the present invention is a method for preparing corneal endothelial cells having a pump function and / or precursor cells thereof capable of differentiating into corneal endothelial cells having a pump function with sufficient proliferation, It is intended to provide a method for producing a corneal endothelial cell sheet for transplantation using the corneal endothelial cell and / or its precursor cell prepared by the method, and the corneal endothelial cell and / or its precursor cell.

The present inventors have found that corneal endothelial progenitor cells can be surprisingly proliferated in vitro by adjusting the serum concentration in the culture of corneal endothelial cells, leading to the present invention. More specifically, by culturing at a serum concentration of 0.5% to 2.0%, corneal endothelial progenitor cells excellent in terms of growth rate and pump function after culture can be prepared.

Accordingly, the present invention relates to the following inventions:
[1] A method for preparing corneal endothelial progenitor cells, comprising culturing corneal endothelial cells or progenitor cells thereof in a culture medium containing 0.5 to 2.0% serum.
[2] The preparation method according to item 1, wherein the culture medium contains basic fibroblast growth factor (bFGF).
[3] The preparation method according to item 1 or 2, wherein the culture medium has a calcium concentration of 50 μM to 200 μM.
[4] The preparation method according to any one of items 1 to 3, wherein the corneal endothelial progenitor cells are cultured in a culture vessel coated with an extracellular matrix molecule.
[5] The preparation method according to item 4, wherein the extracellular matrix molecule is laminin or a functional fragment thereof.
[6] The culture medium is further selected from the group consisting of ascorbyl 2-phosphate, ascorbyl glucoside, ascorbyl ethyl, ascorbyl tetrahexyl decanoate, ascorbyl stearate, and ascorbyl 2-phosphate-6 palmitic acid. 6. The preparation method according to any one of items 1 to 5, comprising a derivative or a combination thereof.
[7] A corneal endothelial progenitor cell that is produced by the preparation method according to any one of items 1 to 6 and has a paving stone shape in which no endothelial mesenchymal transition occurs.
[8] Item 6 wherein the absence of endothelial mesenchymal transition in the corneal endothelial progenitor cells is determined by low level expression of at least one protein selected from the group consisting of α-smooth muscle actin and collagen 1. A corneal endothelial progenitor cell described in 1.
[9] The corneal endothelial progenitor cell according to item 7 or 8, wherein OCT4, Nanog and Lgr5 are positive.
[10] A corneal endothelial cell induced to differentiate from the corneal endothelial progenitor cell according to any one of items 7 to 9.
[11] A method for producing a corneal endothelial cell material supported on a carrier,
Culturing corneal endothelial progenitor cells in a culture medium containing 0.5-2.0% serum;
A method for producing a corneal endothelial cell material, comprising a step of seeding cultured corneal endothelial progenitor cells on a carrier.
[12] The production method according to item 11, comprising a differentiation induction step after the culture.
[13] The production method according to item 11 or 12, wherein in the culturing step, corneal endothelial progenitor cells are cultured in a culture vessel coated with laminin molecules or functional fragments thereof.
[14] The production method according to any one of items 11 to 13, wherein in the culturing step, the calcium concentration of the culture medium is 50 μM to 200 μM.
[15] The production method according to any one of items 11 to 14, wherein, in the culturing step, the culture medium contains basic fibroblast growth factor (bFGF).
[16] The production method according to any one of items 11 to 15, wherein the corneal endothelial cell material is a corneal endothelial cell sheet.

By the method of the present invention, cobblestone-like corneal endothelial progenitor cells can be selectively grown at a high growth rate. The corneal endothelial progenitor cells thus proliferated can differentiate into corneal endothelial cells having a pump function.

FIG. 1A is a photograph showing cell proliferation and shape according to serum concentration. At 0%, almost no cell proliferation was observed, while at 0.5% or more, cell proliferation was observed. In addition, by culturing in the presence of 0.5% serum, cobblestone-shaped cells proliferate, while as the serum concentration increases to 5% and 15%, spindle-shaped cells that are characteristic of fibroblasts Grows. FIG. 1B is a photograph showing the expression levels of αSMA and type 1 collagen, which are indicators of EndMT. While expression was very low at 0% and 0.5%, the expression of αSMA and type 1 collagen increased as the serum concentration increased to 5% and 15%. FIG. 1C is a photograph comparing the culture method of Patent Document 3 with the presence of 1% serum according to the present invention. FIG. 2 shows the results of examining the growth promoting effect by adding various growth factors in the presence of 0.5% serum. When added alone, bFGF has the highest growth promoting effect, and no significant synergistic effect was observed even when various growth factors were added in combination. FIG. 3A is a graph showing a comparison of the extracellular matrix used. By using E8 which is a laminin 511 active fragment, the growth rate was higher than that of atelocollagen. FIG. 3B is a comparative photograph after culture using E8 and atelocollagen in the presence of 1% serum. FIG. 4A is a graph showing the effect of Ca concentration on the growth rate in the presence of serum. FIG. 4B is a graph showing the effect of Ca concentration in the presence of serum on αSMA, which is an EndMT marker, and OCT4, which is an undifferentiated marker. FIG. 5A is a graph showing the effect of Ca concentration on the growth rate after passage in the presence of low serum. FIG. 5B is a graph showing the effect of Ca concentration on αSMA, an EndMT marker, after passage in the presence of low serum. FIG. 5C is a graph showing the effect of Ca concentration on OCT4 which is an undifferentiated marker after passage in the presence of low serum. FIG. 6 is a graph showing the pumping function of corneal endothelial progenitor cells cultured at different serum and calcium concentrations. The photograph shows the result of immunostaining the cell sheet with an anti-Na ⁺ K ⁺ ATPase antibody. FIG. 7A is a graph showing the effect of ascorbic acid 2-phosphate on cell growth rate in low serum low calcium culture. FIG. 7B is a graph showing the effect of ascorbic acid 2-phosphate on αSMA expression as an EndMT marker in low serum low calcium culture.

The present invention relates to a method for preparing corneal endothelial progenitor cells, comprising culturing corneal endothelial progenitor cells in a low serum culture medium.

In the present invention, the corneal endothelial cell is a cobblestone cell located in the innermost layer of the cornea of the eyeball, and may be a cell that has been separated and cultured. The corneal endothelial cells may be collected from living corneal endothelium or may be established corneal endothelial cells. The animal from which the corneal endothelial cells are derived may be any animal, for example, human, monkey, chimpanzee, dog, cat, rabbit, pig, cow, horse, etc. Therefore, as experimental animals, monkeys, chimpanzees and the like are preferable, and humans are most preferable.

Corneal endothelial cells do not have proliferative properties in vivo, and some of the cells collected from living corneal endothelium have proliferative properties. This proliferating cell is referred to as a corneal endothelial progenitor cell in the present invention. Corneal endothelial progenitor cells are cells that have proliferative properties and differentiation properties that differentiate into corneal endothelial cells. In one embodiment, the corneal endothelial progenitor cell can be characterized by the expression of an undifferentiated marker, such as one or more undifferentiated markers selected from the group consisting of Oct4, Nanog, TERT and LGR5. Increased expression can be determined by comparison with expression in corneal endothelial cells or their progenitor cells when cultured at normal serum concentration, ie, 10-15% serum. On the other hand, the expression of p75 used as a marker of corneal endothelial progenitor cells in Non-Patent Document 1 and Patent Document 3 tends to decrease in corneal endothelial progenitor cells prepared according to the present invention. A corneal endothelial progenitor cell that is negative, and is different from a corneal endothelial progenitor cell collected and amplified as a P75-positive cell by FACS in Non-Patent Document 1 according to the present invention. Further, Non-Patent Document 1 describes that p75 positive cells have a neutral crest-like, bipolar spindle shape, and morphologically have corneal endothelial progenitor cells having a cobblestone shape prepared according to the present invention. Can be easily identified. The paving stone-like state refers to a state in which substantially circular cells appear to be spread like the photograph when cultured with 0.5% serum in FIG. 1A. On the other hand, fibroblasts and cells with endothelial mesenchymal transition (EndMT) have a spindle-like shape with polarity as shown in the photograph when cultured with 15% serum in FIG. 1A.

Corneal endothelial cells and their progenitor cells undergo endothelial mesenchymal transition (EndMT) depending on the culture conditions during the culture process. The cells that cause EndMT are not suitable for transplantation as corneal endothelial cells because they lose their pump function. Therefore, it is preferable that the expression of one or more, more preferably all EndMT markers selected from the group consisting of α-smooth muscle actin (αSMA), type 1 collagen (Col1), and fibronectin (FN) is low. On the other hand, even if some of the cultured cells give rise to EndMT, any remaining cells may be cells that have not caused EndMT, so that it is not required that all EndMT markers are completely expressed. Measurement of the expression of the EndMT marker may be performed by a known method known to those skilled in the art. For example, the expressed protein may be measured by an immunoassay technique such as Western blotting, or the amount of mRNA is amplified by PCR. It may be done by doing. In another preferred embodiment, the expression level can be quantified by real-time PCR. The expression of the EndMT marker can determine the level of its expression by comparing it with a high expression level in corneal endothelial cells or its progenitor cells cultured at a normal serum concentration, that is, a serum concentration of 10 to 15% by mass. it can. On the other hand, since the expression of these EndMT markers is also observed at a low level in normal tissue endothelial cells, the expression level equivalent to that of normal tissue endothelial cells can be lowered. Therefore, if the expression of the EndMT marker is equivalent to that of normal tissue endothelial cells, it can be determined that the endMT is not transformed.

In the present invention, low serum means 0.5 to 2.0% by mass of serum. The lower limit of the serum concentration range may be any concentration as long as it can increase the proliferation of corneal endothelial cells or progenitor cells thereof, for example, 0.75% by mass or more, more preferably 1.0% by mass or more. is there. On the other hand, the upper limit of the serum concentration range is 2.0% by mass or less, more preferably 1.5% by mass or less, and still more preferably 1.0% by mass or less from the viewpoint of suppressing EndMT. In general, culture of corneal endothelial cells or their progenitor cells is often performed with 10 to 15% by mass of serum. However, studies by the present inventors have shown that EndMT occurs in some or all cells, although corneal endothelial cells or their progenitor cells increase at a high proliferation rate under 15% by mass of serum ( FIG. 1A). EndMT converted corneal endothelial cells or precursor cells thereof, which are endothelial cells, into mesenchymal cells, and was not suitable as a corneal endothelial cell for transplantation. On the other hand, in the serum-free medium, the proliferation rate of corneal endothelial cells or their progenitor cells was remarkably low and hardly increased (FIG. 1A). Therefore, the above-mentioned low serum concentration range is particularly preferable for the proliferation of corneal endothelial progenitor cells because EndMT is suppressed while maintaining the proliferation of corneal endothelial progenitor cells.

The serum used in the present invention may be any serum, and usually commercially available serum can be used. The origin of serum may be any animal species, and may be serum derived from animals such as humans, monkeys, chimpanzees, dogs, cats, rabbits, pigs, cows, horses, etc., and their fetal serum is used. You can also. As the serum, for example, fetal bovine serum (FBS) can be used, but from the viewpoint of producing cells or cell sheets used for transplantation, human-derived serum can also be used.

In the present invention, a growth factor or a growth factor may be further added in order to enhance the proliferation of corneal endothelial cells or their precursor cells. Growth factors or growth factors that can be added include acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (sometimes referred to as bFGF or FGF2), epidermal growth factor (EGF), hepatocyte growth factor ( HGF), Wnt3A and the like, and these growth factors may be used alone or in combination. bFGF (FGF-2) is known to be a direct mediator that induces endothelial mesenchymal transition (EndMT) in corneal endothelial cells (Non-Patent Document 2: The Journal of Chemistry, Chemistry, Vol. 2004) .279, No.31, pp.32325-32332). On the other hand, when basic fibroblast growth factor (bFGF) is added to a low serum medium, it surprisingly suppresses EndMT and enhances the proliferation of corneal endothelial cells or their progenitor cells. Is particularly preferable (FIGS. 1A and 1B, FIG. 2). From the viewpoint of improving the proliferation rate, it is more preferable to use a combination of bFGF and Wnt3A (FIG. 2B). The concentration of these factors in the cell culture medium can be appropriately selected and selected by those skilled in the art depending on the type of growth factor or growth factor, and is 0.5 to 100 ng / ml, preferably 1 to 50 ng. / Ml, more preferably 2 to 25 ng / ml.

The culture medium used in the present invention may be any medium as long as it is a medium used for cell culture. Examples of the culture medium include Dulbecco's modified Eagle medium (DME), minimum essential medium (MEM), F12, DME / F12, EpiLife (registered trademark) Medium, and the like. The medium used in the present invention may contain additional components such as serum, growth factors and growth factors. As further components contained in the culture medium, amino acids, cell growth promoters, and the like can be appropriately added. On the other hand, it was found that by limiting the concentration of calcium contained in the normal medium, the expression of the undifferentiated marker (OCT4) was increased while suppressing the expression of αSMA, which is the EndMT marker (FIG. 4). And FIG. 5), it is preferable to maintain the calcium concentration at a value lower than 1800 μM used in normal culture, but even if it exceeds 1800 μM, it can be used from the viewpoint of growth rate. The upper limit of the calcium concentration range is 1800 μM or less, preferably 600 μM or less, more preferably 200 μM or less, and even more preferably 150 μM or less from the viewpoint of enhancing the expression of the undifferentiated marker while suppressing the expression of the EndMT marker. . The lower limit of the calcium concentration range is preferably 20 μM or more, more preferably 40 μM or more, further preferably 50 μM or more, and even more preferably 60 μM or more, from the viewpoint of enabling cell growth.

The culture medium used in the present invention may further contain an ascorbic acid derivative. Ascorbic acid derivatives can suppress EndMT while promoting proliferation of corneal endothelial cells and / or their progenitor cells. The derivatives of ascorbic acid are not intended to be limited to the following, but include, for example, ascorbic acid 2-phosphate, ascorbyl glucoside, ascorbyl ethyl, ascorbyl tetrahexyldecanoate, ascorbyl stearate, ascorbic acid- A diphosphate-6 palmitic acid is mentioned. Ascorbic acid derivatives may be used alone or in combination. Ascorbic acid may be in the L form, D form, or racemic form, but the L form is preferred from the viewpoint of having physiological activity. The concentration of the ascorbic acid derivative is preferably 10 μg / ml or more, more preferably 30 μg / ml or more, and even more preferably 50 μg / ml or more from the viewpoint of promoting cell growth and / or suppressing EndMT. From the viewpoint of suppressing the pH change, 1000 μg / ml or less is preferable, 500 μg / ml or less is preferable, and 200 μg / ml or less is more preferable.

The low serum culture medium of the present invention includes growth factors, growth factors, low concentrations of Ca, and ascorbic acid or derivatives thereof from the viewpoint of suppressing EndMT while promoting proliferation of corneal endothelial cells and / or their precursor cells. One or more of these are added. All of these additional components except serum are preferably combined.

Corneal endothelial cells or their progenitor cells can be cultured by adhering them to an arbitrary culture vessel. The culture container to be used may be coated with a biocompatible polymer. The biocompatible polymer used is one or more molecules selected from extracellular matrix molecules such as laminin, collagen, elastin, fibronectin, fibrinogen, thrombospodin, gelatin, heparan sulfate, chondroitin sulfate, or the like. It may be a functional fragment thereof. From the viewpoint of ensuring cell proliferation, laminin, collagen, or a functional fragment thereof is particularly preferable, and laminin or a functional fragment thereof is most preferably used. The functional fragment refers to a fragment of the protein that controls the function of the protein, and includes modified forms thereof.

Laminin is composed of heterotrimers of α chain, β chain, and γ chain. There are 5 types of α chains, 4 types of β chains, and 3 types of γ chains as a family, but any combination may be used. In particular, from the viewpoint of ensuring the proliferation of corneal endothelial progenitor cells, laminin 511, 521, 332 or a functional fragment thereof is preferred, and laminin 511 or a functional fragment thereof is most preferred.

As the culture conditions of the method for preparing corneal endothelial precursor cells of the present invention, any conditions used for culturing animal cells can be used. For example, in the case of mammalian cells, the cells may be cultured at 35 to 39 ° C., more preferably 36 to 38 ° C., even more preferably 37 ° C. in an atmosphere of 2 to 15%, preferably 5% CO _2. However, it can usually be cultured in an incubator set to the above conditions.

Corneal endothelial progenitor cells prepared according to the present invention can be differentiated into corneal endothelial cells by further induction of differentiation. As a method for inducing differentiation, any means may be used. For example, differentiation can be induced by maintaining cells in a confluent state for about two weeks. In one embodiment, differentiation can be induced by seeding in a DME medium containing 10 to 15% FBS at a cell density of 1000 cells / mm ² or more and culturing for 2 to 3 weeks. On the other hand, if high-concentration FBS is used as described above, there is a possibility of inducing EndMT. Therefore, as a result of seeding at a high density of 1000 cells / mm ² or more using 1-2% low-concentration FBS and culturing for 2-3 weeks, it was confirmed that differentiation could be sufficiently induced even at low FBS concentrations. ing. As a method for confirming differentiation induction, it can be confirmed by observing the expression of Na ⁺ K ⁺ ATPase, which plays an important role in the pump function of corneal endothelial cells, in the periphery of the cell by immunostaining. Moreover, it can confirm by actually measuring the presence or absence of the pump function of a cell by the method explained in full detail in an Example.

The corneal endothelial progenitor cells prepared according to the present invention and / or corneal endothelial cells obtained by inducing differentiation of the progenitor cells can be used for the treatment of diseases caused by corneal endothelial cell damage. More specifically, corneal endothelial progenitor cells prepared according to the present invention and / or corneal endothelial cells obtained by inducing differentiation of the progenitor cells can be injected into the anterior chamber and directly fixed on the corneal endothelium. In addition, the corneal endothelial cell material supported on the carrier may be fixed on the corneal endothelium. Therefore, the present invention also relates to a method or composition for treating a disease caused by damage to corneal endothelial cells.

In the present invention, the corneal endothelial cell material refers to a material for transplantation in which corneal endothelial cells or their precursor cells are supported on a carrier. Any carrier can be used as long as it can be used as a cell carrier. For example, by using a sheet-like carrier, the corneal endothelial cell material can be used as a corneal endothelial cell sheet. More preferably, the cell carrier is preferably a material that is absorbed into the living body and disappears after transplantation. For example, a cell carrier obtained by molding an extracellular matrix such as collagen, gelatin, or vitrigel into a sheet shape can be used.

The corneal endothelial cell material can be produced by a production method including a step of culturing corneal endothelial progenitor cells and a step of seeding the cultured corneal endothelial progenitor cells on a carrier. The step of culturing corneal endothelial progenitor cells refers to the method for culturing corneal endothelial cells according to the present invention. This production method may further include a step of inducing differentiation of corneal endothelial progenitor cells. Differentiation induction may be performed before seeding on a carrier or after seeding.

Method for obtaining human corneal endothelial cell preparation Advanced DME containing 100 μg / ml ascorbic acid-2-phosphate (Wako Pure Chemicals), 2 ng / ml bFGF (Wakken), 1 × Glutamax (Gibco) After washing with / F12 medium (Gibco) (hereinafter referred to as basal medium), the corneal endothelium was peeled off together with the Descemet's membrane using a fine scissors, and transferred to a 35 mm Petri dish. The Descemet membrane piece to which the corneal endothelial cells adhered on the Petri dish was further cut into small pieces of about 5 mm square and collected in a centrifuge tube. The supernatant was removed and 1 mL of basal medium containing 0.2% collagenase (Roche) was added and incubated at 37 ° C., 5% CO ₂ for 1-3 hours. The supernatant was removed, 10 mL of PBS was added, and the mixture was washed by centrifugation at 500 g for 5 minutes. 1 mL of 0.5% trypsin / 0.2% EDTA was added to the cell mass, and the cells were dispersed by pipetting after incubation at 37 ° C. and 5% CO ₂ for 5 minutes. 10 mL of basal medium was added, centrifuged at 500 g for 5 minutes, the supernatant was removed, and the resulting cell pellet was suspended in 1 mL of basal medium (hereinafter referred to as human corneal endothelial cell preparation).

Examination of fetal bovine serum (FBS) concentration Add 1 ml of 50 μg / mL atelocollagen solution (Koken) diluted with 10 mM acetic acid to a 6-well dish, leave at 37 ° C. for 1 hour, and then wash twice with 5 ml of PBS to wash atelocollagen. A coat dish was prepared. Human corneal endothelial cell preparations were evenly suspended in a basal medium containing 0%, 0.5%, 5%, 15% FBS (Nichirei). Each cell suspension was seeded on an atelocollagen-coated dish and cultured at 37 ° C. under 5% CO ₂ for 2-3 weeks while changing the medium every 2-3 days. FIG. 1A shows a cell image after 2 weeks of culture. In the 0% FBS group, almost no cell proliferation was observed, but in the 0.5% FBS group, papillary cell proliferation similar to that of the corneal endothelium was observed. On the other hand, in the groups using 5% and 15% FBS, although cell proliferation was observed, the cell morphology was changed to fibroblast-like.

RT-PCR
When the cells became sub-confluent, the cells were detached and accumulated with the above trypsin / EDTA, and used as a sample for RNA extraction. For RNA extraction, RNeasy mini kit (Qiagen) was used, and concentration measurement and quality check were performed with Nanodrop. Using an RNA equivalent to 500 ng as a template, cDNA was synthesized using Advantage (registered trademark) cDNA PCR Kit (Clontech). The synthesized cDNA was diluted to 1/5 and used as a sample for PCR. The primers listed in Table 1 below were used. Titanium taqDNA polymerase (Clontech) was used according to the attached reaction conditions, and amplified at 30 ° C to 35 ° C at 94 ° C, 63 ° C and 72 ° C. Electrophoresis was performed on a 2% agarose gel, and the expression levels were compared (FIG. 1B). As a result, in the groups using 5% and 15% FBS, enhanced expression of αSMA and Type1 collagen, which are transformation markers, was confirmed.

Comparison with known culture methods A human corneal endothelial cell preparation was obtained by the following method disclosed in Patent Document 3. 3 ml of 20 μg / ml laminin 511 (Veritas) diluted with PBS was added to a 10 cm dish, allowed to stand at 37 ° C. for 3 hours, and then washed twice with 5 ml PBS to prepare a laminin 511 coated dish. The Descemet's membrane piece with corneal endothelial cells attached was transferred to a centrifuge tube containing DME (Gibco) containing 10 μM Y-27632 (Wako Pure Chemical Industries), and left at 37 ° C. for 30 minutes. The supernatant was removed, 1 mL of Stem Pro Accutase (Invitrogen) was added, and the mixture was treated at 37 ° C. for 30 minutes. 4 mL of DME was added, and after centrifugation at 1500 rpm for 5 minutes, the supernatant was removed and the cell pellet was used as a human corneal endothelial cell preparation. Basal medium containing 0.5% FBS, which is the medium of the present invention, and the medium disclosed in Patent Document 3, that is, 20% KnockOUT Serum Replacement (Invitrogen), 2 mM L-glutamine, 1% non-essential amino acid, 100 μM 2 -DMEM / F-12 medium (Invitrogen) containing mercaptoethanol, 4 ng / ml bFGF was prepared. The above human corneal endothelial cell preparation was evenly suspended in these media and seeded on laminin 511 coat dishes at a density of 500 cells / cm2. The culture medium was changed every 2 to 3 days in each medium and cultured for 3 weeks. As a result, when the culture solution of the present invention was used, paving stone cells proliferated actively and reached confluence in 3 weeks. On the other hand, in the culture solution of Patent Document 3, the growth of a neural crest-like bipolar spindle-shaped cell similar to that shown in Non-Patent Document 1 was confirmed, but the growth stopped without reaching confluence (FIG. 1C). . As a result of undifferentiated marker expression analysis, as shown in the upper part of the photograph, expression of OCT4 and Lgr5 was observed in the culture of the present invention, but expression of Pax6 and p75 was not recognized. In addition, the culture method of Patent Document 3 could not perform expression analysis because cell proliferation was insufficient.

Examination of growth factors to be added 0.5% FBS (Nichirei) was added to a basal medium not containing FGF, and various growth factors were further added to prepare the following medium. (1) Growth factor-free control, (2) 2 ng / ml bFGF, (3) 50 ng / ml RSPO1 (R & D), (4) 50 ng / ml Wnt3A (R & D), (5) 2 ng / ml bFGF + 50 ng / ml RSPO1, (6) 2 ng / ml bFGF + 50 ng / ml Wnt3A, (7) 50 ng / ml RSPO1 + 50 ng / ml Wnt3A, (8) 2 ng / ml bFGF + 50 ng / ml RSPO1 + 50 ng / ml Wnt3A. Human corneal endothelial cell preparations suspended in a basal medium without bFGF are evenly suspended in these mediums, and each cell suspension is seeded on an atelocollagen-coated dish at 37 ° C. under 5% CO ₂ . The culture was performed every 2 to 3 days while changing the medium. After 12 days, the cells were detached with trypsin / EDTA and the number of cells was counted. As a result, bFGF had the highest growth promoting effect when the growth factor alone was added, and the combination of bFGF and Wnt3A showed an additive growth promoting effect. However, even when various growth factors were added in combination, remarkable synergy was observed. No effect was observed (FIG. 2).

Effect of laminin 511 1 ml of 10 μg / ml E8 diluted with PBS (iMatrix-511, product expressing only the active site of laminin 511, Nippi) was added to a 6-well dish and left at 37 ° C. for 3 hours. An E8 coat dish was prepared by washing twice with PBS. For comparison, an atelocollagen-coated dish prepared by adding 1 ml of a 50 μg / mL atelocollagen solution (Koken) diluted with 10 mM acetic acid to a 6-well dish, leaving it at 37 ° C. for 1 hour, and then washing twice with 5 ml of PBS. Using. The human corneal endothelial cell preparation was evenly suspended in a basal medium containing 0.5%, 1%, 2%, 5% FBS (Nichirei) and seeded in a 6-well dish coated with E8 or atelocollagen. The medium was changed every 2-3 days with each medium, and after 11 days, the cells were detached with trypsin / EDTA and the number of cells was counted (FIG. 3A). As a result, in the E8 coat group, the growth rate was highest when the serum concentration was 1%, and the morphology showed a paving stone characteristic of corneal endothelial cells. On the other hand, the atelocollagen-coated group showed serum concentration-dependent cell growth, but the cell growth was lower than that of E8, and particularly little growth was observed in the low concentration FBS group of 0.5% to 2%. . FIG. 3B shows cell images in the 1% FBS group on E8 and atelocollagen.

Effect of low-concentration Ca medium LN511 (Veritas) diluted with PBS to 10 μg / ml was added to a 6-well dish, allowed to stand at 37 ° C. for 3 hours, and then washed twice with 5 ml of PBS to prepare an LN511-coated dish. EpiLife® Medium containing 2 ng / ml bFGF (containing 60 μM CaCl ₂ .2H ₂ O) (Gibco) (hereinafter referred to as low Ca medium) and 1800 μM, which is the concentration at which calcium concentration is normally used for low Ca medium A medium containing 1%, 2%, 5%, and 10% FBS was prepared in each of the media to which CaCl ₂ .2H ₂ O was added (hereinafter referred to as normal Ca media). The human corneal endothelial cell preparation obtained by the method for obtaining the human corneal endothelial cell preparation described above was evenly suspended in each medium, and seeded in a 6-well dish coated with LN511. The medium was changed every 2-3 days with each medium, and after 10 days, the cells were detached with trypsin / EDTA and the number of cells was counted. FIG. 4A shows the results of cell proliferation. In normal Ca medium, the cell growth rate was the fastest in the 1% FBS group. On the other hand, the growth rate was the fastest in the 2% FBS group in the low Ca medium, and the growth rate was slightly reduced in the 1% FBS. In any group, similar to the results shown by the effect of laminin 511, the low serum culture showed higher growth than the culture at the 10% serum concentration that is usually used.

Real-time PCR
Trypsinized cells were used as samples for RNA extraction. RNA extraction was carried out by isopropanol precipitation and purification using TRIzol (registered trademark) (Ambion). The extracted RNA was subjected to concentration measurement and quality check using Nanodrop. CDNA equivalent to 500 ng of RNA was synthesized using Advantage (registered trademark) cDNA PCR Kit (Clontech). Synthetic cDNA was diluted to 1/5 and used as a sample for RT-PCR. RT-PCR was performed using Thermal Cycler Dice (registered trademark) Real Time System II (TaKaRa). As a reaction reagent, SYBR (registered trademark) Premix Ex Taq (trademark) II was used according to the attached reaction conditions. The primers listed in Table 2 below were used. As a result, the expression of αSMA, a transformation marker, was found to be increased depending on the FBS concentration. In general, the low Ca culture showed a lower value than the normal Ca culture, and the low Ca medium 1% FBS group had the lowest value. (FIG. 4B). On the other hand, the expression of OCT4, an undifferentiated cell marker, was higher in the low Ca medium group than in the normal Ca medium group, particularly in the low-concentration FBS group, and increased in the low-Ca medium 1% FBS group. (FIG. 4C). From these results, it was suggested that low Ca culture has an effect of suppressing transformation and maintaining undifferentiation. From the viewpoint of cell proliferation, 2% FBS was superior to 1% FBS, but 1% FBS was considered more useful in terms of transformation inhibition and maintenance of undifferentiation.

Evaluation of cell stability after passage 3 mL of LN511 (Veritas) diluted to 10 μg / ml with PBS was added to a 10 cm dish, left at 37 ° C. for 3 hours, and then washed twice with 5 ml of PBS to prepare an LN511 coated dish. did. A medium in which 2% FCS was added to each of the low Ca medium and the normal Ca medium was prepared. The human corneal endothelial cell preparation obtained by the above-described method for obtaining the human corneal endothelial cell preparation was evenly suspended in each of the above media and seeded in a 10 cm dish coated with LN511. Each medium was cultured for about 2 weeks while changing the medium every 2-3 days.

When the cells became sub-confluent, the cells were detached with trypsin EDTA, centrifuged and washed, suspended in each medium at a concentration of 1000 cells / cm ² , and seeded on LN511 coated dishes. When the cells became subconfluent, the passage operation was repeated in the same manner to evaluate the cell stability (FIG. 5A). As a result, regarding the cell growth rate, the tendency for the growth rate to increase at each passage was recognized, and the growth of the low Ca culture was slightly faster than that of the normal Ca culture. ΑSMA expression of the transformation marker was lower in the low Ca culture than in the normal Ca culture, and in the normal Ca medium group, the αSMA expression increased at each passage, but in the low Ca medium group, the low αSMA expression was low after the passage. Levels were maintained (Figure 5B). On the other hand, the expression of OCT4, an undifferentiated cell marker, was higher in the low Ca medium group than in the normal Ca medium group, and the low Ca medium group maintained a high expression level after passage (FIG. 5C). From these results, it was suggested that low Ca culture also has the effect of suppressing transformation and maintaining undifferentiation in 2% FBS culture.

A human corneal endothelial cell preparation obtained by the method for obtaining a human corneal endothelial cell preparation described in the preparation of a cell sheet was prepared by using a low Ca medium containing 2% FBS, a normal Ca medium, and a normal Ca medium containing 15% FBS. The suspension was evenly suspended in each of these and seeded in a 10 cm dish coated with LN511. While changing the medium every 2-3 days in each medium, the cells were cultured for about 2-3 weeks until the cells became subconfluent. The cells were detached with trypsin EDTA, centrifuged, washed, suspended in DME medium (differentiation medium) containing 15% FBS, 2 ng / ml FGF, and swollen with PBS at 3000 cells / mm ^2. Each cell was seeded at a concentration. Cell sheets were produced by changing the differentiation medium every 3-4 days and culturing for 14 days.

Cell sheet immunostaining The cell sheet was cut out with a 6 mm trepan, placed on a 24-well plate, 2 mL of cold methanol was added, and fixed in a freezer for 10 minutes. After removing methanol with an aspirator, it was air-dried and washed with 1 mL of PBS (hereinafter referred to as a washing buffer) containing 0.15% triton (Sigma). The cell sheet was blocked with PBS containing 250 μL of 3% BSA (Sigma) and 0.3% triton at room temperature for 30 minutes. The blocking buffer was removed with an aspirator, 250 μL of Na ⁺ K ⁺ ATPase antibody (Merck Millipore) diluted 200-fold with a washing buffer was added, and reacted at room temperature for 2 hours. The antibody solution was removed with an aspirator and washed 3 times with 1 mL of washing buffer. 250 μL of Alexafluoro-labeled anti-mouse antibody (Life Technology) diluted 200-fold with a washing buffer was added and reacted at room temperature for 1 hour under light shielding. The antibody solution was removed with an aspirator and washed 3 times with 1 mL wash buffer. The encapsulant was placed on the cell sheet, covered with a cover glass, and observed with a fluorescence microscope. FIG. 6 shows the results of immunostaining the cell sheet produced by each method with an anti-Na ⁺ K ⁺ ATPase antibody. In each cell sheet, it was confirmed that Na ⁺ K ⁺ ATPase was differentiated into corneal endothelial cells from the localization of the cell periphery.

Measurement of pump function Using a Wushing chamber system (WPI), the pump function of cells was measured by the following method. A voltage electrode was prepared with 2% agarose 3M KCl and set in a chamber. The DMEM medium containing 5% FBS and 2 ng / ml FGF was evenly filled in the left and right chambers, and the medium was equilibrated by bubbling a mixed gas of 95% air and 5% CO ₂ . A cell sheet placed on the slider was filled into the chamber, and voltage measurement was started. After confirming that the voltage was stable for about 10 minutes, ouabain was added to a final concentration of 0.5 mM, and the potential difference before and after the addition was measured (FIG. 6). As a result, no potential difference could be confirmed in cells amplified and cultured in 15% FBS normal Ca medium. On the other hand, a potential difference of about 3 μV was observed in cells amplified and cultured in 2% FBS normal Ca medium. Furthermore, a potential difference of about 10 μV was observed in cells amplified and cultured in 2% FBS low Ca medium. From this, it was found that cells having a high pump function can be obtained by amplifying the cells in a low serum medium and a low serum low Ca medium.

Effect of ascorbic acid 2-phosphate in low serum low-concentration Ca medium 1 mL of LN511 (Veritas) diluted to 10 μg / ml with PBS was added to a 6-well dish, left at 37 ° C. for 3 hours, and then twice with 5 ml of PBS. Washed to prepare an LN511 coated dish. EpiLife (registered trademark) Medium (containing 60 μM CaCl ₂ · 2H ₂ O) (Gibco) (hereinafter referred to as low Ca medium) containing ₂ ng / ml bFGF and 1% FBS at a final concentration of 100 μg / ml ascorbic acid A medium containing -2 phosphate and an ascorbic acid-2 phosphate-free medium were prepared. Human corneal endothelial cell preparations obtained according to the above-described method for obtaining human corneal endothelial cell preparations were evenly suspended in each medium, and seeded at 10000 cells / well in a 6-well dish coated with LN511. The medium was changed every 2-3 days with each medium, and after 10 days, the cells were detached with trypsin / EDTA and the number of cells was counted. FIG. 7A shows the result of the number of cells counted. Cells cultured in a low serum low-concentration Ca medium containing ascorbic acid-2 phosphate showed a faster cell growth rate than a medium not containing ascorbic acid-2 phosphate.

Tripsinized cells were used as samples for RNA extraction, real-time PCR was performed according to the method described above, and the expression level of GAPDH as αSMA and an internal standard was measured. As a result, as shown in FIG. 8B, αSMA expression of the transformation marker was lower in the medium containing ascorbic acid-2 phosphate than in the medium not containing it.

Claims (16)

A method for preparing corneal endothelial progenitor cells, comprising culturing corneal endothelial cells or progenitor cells thereof in a culture medium containing 0.5 to 2.0% serum. The preparation method according to claim 1, wherein the culture medium contains basic fibroblast growth factor (bFGF). The preparation method according to claim 1 or 2, wherein the culture medium has a calcium concentration of 50 µM to 200 µM. The preparation method according to any one of claims 1 to 3, wherein the corneal endothelial progenitor cells are cultured in a culture vessel coated with extracellular matrix molecules. The preparation method according to claim 4, wherein the extracellular matrix molecule is laminin or a functional fragment thereof. An ascorbic acid derivative selected from the group consisting of the ascorbic acid 2-phosphate, ascorbyl glucoside, ascorbyl ethyl, ascorbyl tetrahexyl decanoate, ascorbyl stearate, and ascorbic acid-2-phosphate-6 palmitic acid; Or the preparation method according to any one of claims 1 to 5, comprising a combination thereof. A corneal endothelial progenitor cell that is produced by the preparation method according to any one of claims 1 to 6 and exhibits a paving stone shape in which no endothelial mesenchymal transition occurs. The absence of endothelial mesenchymal transition in the corneal endothelial progenitor cells is determined by low level expression of at least one protein selected from the group consisting of α-smooth muscle actin and collagen 1. Corneal endothelial progenitor cells. The corneal endothelial progenitor cell according to claim 7 or 8, wherein OCT4, Nanog and Lgr5 are positive. A corneal endothelial cell induced to differentiate from the corneal endothelial progenitor cell according to any one of claims 7 to 9. A method for producing a corneal endothelial cell material carried on a carrier,
Culturing corneal endothelial progenitor cells in a culture medium containing 0.5-2.0% serum;
A method for producing a corneal endothelial cell material, comprising a step of seeding cultured corneal endothelial progenitor cells on a carrier. The production method according to claim 11, comprising a differentiation induction step after the culture. The production method according to claim 11 or 12, wherein in the culturing step, corneal endothelial progenitor cells are cultured in a culture container coated with laminin molecules or functional fragments thereof. The production method according to any one of claims 11 to 13, wherein in the culturing step, the calcium concentration of the culture medium is 50 μM to 200 μM. The production method according to any one of claims 11 to 14, wherein, in the culturing step, the culture medium contains basic fibroblast growth factor (bFGF). The production method according to any one of claims 11 to 15, wherein the corneal endothelial cell material is a corneal endothelial cell sheet.

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