JP2017099381A - Cell sheet comprising amniotic fluid stem cell and method for producing the same - Google Patents

Abstract

A technique for applying amniotic fluid stem cells to surgical treatment and various skin care is provided.
A cell sheet containing amniotic fluid stem cells.
[Selection] Figure 4

Description

  The present invention relates to a cell sheet containing amniotic fluid stem cells and a method for producing the same.

  Amniotic fluid is a fluid that fills the egg membrane from the beginning of pregnancy to delivery, and is no longer needed for delivery from the mother to the body before and after fetal delivery. The fetus floats and grows in amniotic fluid. Amniotic fluid is thought to be constantly generated from the fetus and placenta, but details are still unknown. In the amniotic fluid test performed in the second trimester, amniotic fluid is collected by amniocentesis. At this time, the decrease in amniotic fluid due to collection does not affect the fetus, and the amniotic fluid is regenerated and replenished.

  Amniotic fluid contains cells called amniotic fluid cells. Amniotic fluid cells are cells derived from fetal organs during the embryonic period. Non-Patent Document 1 describes that c-Kit (CD117) positive cells were obtained from human amniotic fluid cells.

Pozzobon M., et al., Isolation of c-Kit + Human Amniotic Fluid Stem Cells from Second Trimester, Methods in molecular biology, 1035, 191-198, 2013.

  Since amniotic fluid cells are juvenile, they are considered to have excellent ability to divide and regenerate, have plasticity, and have high immune tolerance. Then, an object of this invention is to provide the technique which applies amniotic fluid stem cells to the surgical treatment and skin care which do not depend on HLA type | mold.

The present invention includes the following aspects.
(1) A cell sheet containing amniotic fluid stem cells.
(2) The cell sheet according to (1), which is used for autologous transplantation or allogeneic transplantation.
(3) The cell sheet according to (1) or (2), wherein the amniotic fluid stem cells are CD117 positive cells.
(4) The cell sheet according to any one of (1) to (3), wherein the cell density is 3 × 10 ⁵ cells / cm ² or more.
(5) a step of recovering CD117-positive amniotic fluid cells, a step of seeding the recovered cells on a culture substrate at a density of 3 × 10 ⁵ cells / cm ² or more and culturing in a sheet form, and the culture substrate And a step of separating and collecting the cells in a sheet form.
(6) The production method according to (5), wherein the culture substrate is a culture substrate having a surface coated with a temperature-responsive polymer.
(7) A cell sheet produced by the production method according to (5) or (6).

  ADVANTAGE OF THE INVENTION According to this invention, the technique which applies amniotic fluid stem cells to the surgical treatment and skin care which do not depend on HLA type | mold can be provided.

(A)-(c) are the optical microscope photographs of the amniotic fluid cell after isolate | separating with CD117 antibody in Experimental example 2. FIG. (A) is a photograph of 4 times magnification, (b) is a photograph of 10 times magnification, and (c) is a photograph of 20 times magnification. (A)-(h) is a graph which shows the result of having analyzed the surface antigen marker of CD117 positive amniotic fluid cell by the flow cytometry in Experimental example 2. FIG. (A) is a photograph showing the results of confirming that CD117-positive amniotic fluid cells were induced to differentiate into bone and differentiated into bone by Alizarin Red S staining in Experimental Example 2. The magnification is 20 times. (B) is a photograph showing the result of confirming that CD117-positive amniotic fluid cells were induced to differentiate into adipose tissue and differentiated into fat by Oil Red staining in Experimental Example 2. The magnification is 20 times. (C) is a photograph showing the results of confirming that CD117-positive amniotic fluid cells were induced to differentiate into cartilage tissue and differentiated into cartilage tissue by Alcian Blue staining and hematoxylin-eosin (HE) staining in Experimental Example 2. The magnification is 40 times. 6 is a photograph of a cell sheet produced in Experimental Example 3. (A) is a photograph showing the result of HE staining of a cell sheet in Experimental Example 4. (B) is a photograph showing the result of staining a cell sheet with an anti-vimentin antibody in Experimental Example 4. (A) is a representative photograph of the wound of the control group in Experimental Example 5. Further, (b) is a representative photograph of the wound of the treatment group in Experimental Example 5. It is a graph which shows the change of the wound area of the control group in Example 5, and a treatment group. (A) And (b) is a photograph which shows the result of the Picrosirius red dyeing | staining of the wound part of Experimental Example 5 on the 21st day from wound preparation.

[Cell sheet]
In one embodiment, the present invention provides a cell sheet comprising amniotic fluid stem cells. The cell sheet of this embodiment includes amniotic fluid stem cells in the cells constituting the cell sheet. Amniotic fluid stem cells can be collected in a minimally invasive manner, and even if collected, there are few ethical problems when used as therapeutic stem cells.

  Examples of amniotic fluid stem cells include CD117 positive cells. As will be described later in the Examples, CD117-positive amniotic fluid stem cells have been confirmed to have the ability to differentiate into bone, cartilage, adipose tissue, etc., and are useful as therapeutic stem cells. Amniotic fluid stem cells are present in amniotic fluid, and even if a certain amount of amniotic fluid is collected during pregnancy, it can be naturally regenerated and supplemented, so that amniotic fluid stem cells can be collected multiple times. Therefore, it is possible to secure a sufficient amount of therapeutic cells before birth for a fetus expected to be treated by this method after birth. In Japan, about 20,000 amniotic fluid tests are conducted annually for fetal chromosome testing, and when the normal karyotype is found and childbirth is completed successfully, the cultured amniotic fluid cells are discarded as medical waste. Has been. For this reason, banking like a cord blood bank is also realistic.

  The cell sheet of the present embodiment can be produced by a cell sheet production method described later, and is obtained by culturing amniotic fluid stem cells in a sheet form. Therefore, the cell sheet of the present embodiment can be referred to as “a cell sheet produced from amniotic fluid stem cells” or “a cell sheet obtained by culturing amniotic fluid stem cells into a sheet”.

  Moreover, the sheet | seat which consists of a cell from which the amniotic fluid stem cell differentiated in the process of culture | cultivating an amniotic fluid stem cell in the sheet form, and the property as a stem cell was lost is also included by this invention. Thus, in the cell sheet of this embodiment, it is preferable that at least a part of the cells constituting the cell sheet are amniotic fluid stem cells, but they may be differentiated cells.

  Since the cell sheet of this embodiment has a sheet-like shape, it can be easily used for surgical treatment or the like. For example, the cell sheet of this embodiment can be used for the treatment of congenital fetal diseases diagnosed by ultrasonic diagnosis or the like before birth. Examples of such congenital diseases include cleft palate, cleft lip, congenital heart disease, umbilical hernia, abdominal wall rupture, and spinal meningostomy. The cell sheet according to the present embodiment can be used for organ protection before treatment of these diseases, wound healing promotion after treatment, keloid treatment, various skin care including anti-aging, and the like.

  In addition, since the cell sheet of this embodiment can be prepared in advance from amniotic fluid collected by amniocentesis before birth, it can be used for treatment by autologous transplantation immediately after birth. In addition, since the cell sheet of the present embodiment has high immune tolerance, it is considered that there is little rejection even when used not only for autotransplantation but also for allogeneic transplantation.

The cell sheet of this embodiment preferably has a cell density of 3 × 10 ⁵ cells / cm ² or more. As will be described later in Examples, a cell sheet having a cell density in the above range has a strength sufficient to maintain its shape, and is easy to handle and easy to apply.

[Method for producing cell sheet]
In one embodiment, the present invention includes a step of recovering CD117-positive amniotic fluid cells, and a step of seeding the recovered cells on a culture substrate at a density of 3 × 10 ⁵ cells / cm ² or more and culturing in a sheet form. And a step of peeling and collecting the cells from the culture substrate in a sheet form. Unlike normal stem cells, the amniotic fluid stem cells of the present invention are obtained by substituting and substituting adherent cells in a non-adhesive culture dish when cells are collected from amniotic fluid. Surprisingly, when seeded on a plastic dish at a high concentration of about 3 × 10 ⁵ cells / cm ^2, a cell sheet composed of stem cells having multipotency could be prepared in a short period of 3 days.

(Step of collecting CD117-positive amniotic fluid cells)
In this step, CD117 positive cells are collected from the amniotic fluid cells. As the amniotic fluid cells, cells contained in amniotic fluid collected by amniocentesis can be used.

  CD117 positive cells may be collected immediately after collection of amniotic fluid. However, since the number of amniotic fluid cells contained in the amniotic fluid is small, it is better to collect CD117 positive cells after the amniotic fluid cells are cultured to some extent and proliferated. It's easy to do.

  For the growth of amniotic fluid cells, it is preferable to use an uncoated culture substrate that is not coated with a cell adhesion factor or the like, and cells that adhere to the culture substrate are used. Examples of the material for the culture substrate include polystyrene and glass.

  The recovery of CD117-positive amniotic fluid cells can be performed by separation using a cell sorter using an antibody against CD117, separation using a magnetic bead and a magnetic stand, or the like. The collected CD117-positive amniotic fluid cells may be cultured again and grown. However, from the viewpoint of suppressing the differentiation of CD117-positive amniotic fluid cells (amniotic fluid stem cells), it is preferable to minimize the cell culture after collecting the CD117-positive amniotic fluid cells.

(Process of culturing in sheet form)
In this step, the collected CD117-positive amniotic fluid cells are cultured in a sheet form. For example, CD117-positive amniotic fluid cells can be cultured in a sheet form by seeding and cultivating CD117-positive amniotic fluid cells to a certain degree of density on a culture substrate.

As will be described later in the examples, when the CD117-positive amniotic fluid cells are seeded on a culture substrate at a density of 3 to 6 × 10 ⁵ cells / cm ² or more and cultured in a sheet shape, the sheet shape can be maintained sufficiently. A cell sheet having a sufficient strength can be formed. The upper limit of the cell density of CD117 positive amniotic fluid cells to be seeded is not particularly limited. Examples of the culture time required for forming the cell sheet include, but are not limited to, about 3 days.

(Process to peel and collect cells into a sheet)
In this step, CD117-positive amniotic fluid cells cultured in a sheet form are peeled from the culture substrate. CD117-positive amniotic fluid cells are preferably detached in a sheet form without causing physical damage.

  For example, using a culture substrate coated with a temperature-responsive polymer on the surface as a culture substrate, and culturing CD117-positive amniotic fluid cells in a sheet form on the culture substrate, the cells are physically damaged. Without peeling off into a sheet.

  Although it does not restrict | limit especially as a temperature-responsive polymer, Poly-N-isopropyl acrylamide (PIPAAm) etc. are mentioned. As a culture substrate coated with a temperature-responsive polymer, a commercially available one may be used. In the case of a culture substrate coated with poly-N-isopropylacrylamide, the substrate surface reversibly changes to hydrophobic and hydrophilic at 32 ° C. Therefore, after culturing CD117-positive amniotic fluid cells at 37 ° C. in a sheet form, the cells are physically damaged by incubating the culture substrate at a temperature below 32 ° C., for example, at 20 to 25 ° C. for about 15 minutes. And can be peeled into a sheet. The detached cell sheet can be collected and used for surgical treatment or the like.

  Next, although an experiment example is shown and this invention is demonstrated in detail, this invention is not limited to the following experiment examples.

[Experimental Example 1]
(Culture of human amniotic fluid cells)
Human amniotic fluid cells were collected and cultured. As the human amniotic fluid, the human amniotic fluid of the patient who obtained the consent among the patients who performed the amniotic fluid test at the Keio University Hospital for the purpose of chromosome examination with the approval of the Keio University Hospital Ethics Committee was used. The amniotic fluid test was carried out from 15 weeks of gestation to 17 weeks of gestation, and the sample amount of collected amniotic fluid was 5 to 10 mL. The number of amniotic fluid cells in the amniotic fluid was 1-2 × 10 ⁴ / mL.

  Human amniotic fluid was centrifuged, the supernatant was removed, and then seeded on a 35 mm dish with an uncoated cover glass and cultured at 37 ° C. The medium used was Chang Medium C Lyophilized Kit (Irvine Scientific) supplemented with 2 mM penicillin and streptomycin, 2 mM L-glutamine.

  The amount of the medium was 2 mL / 35 mm dish. The medium was changed on days 5 to 7, and the first passage was performed on days 10 to 12. After the first passage, the medium was changed to Expansion medium and cultured in a plastic 100 mm dish. The passage was repeated when the cells were 80-90% confluent.

  The composition of the expansion medium was 20% Chang Medium, 15% fetal calf serum (bioest), 2 mM penicillin and streptomyces, 2 mM L-glutamine in MEM Alpha (Gibco) medium.

[Experiment 2]
(Analysis of CD117 positive cells)
In Experimental Example 1, CD117 positive cells were separated from human amniotic fluid cells after passage 2 times after changing to the expansion medium, and analyzed.

  More specifically, first, CD117 positive cells using a commercially available kit (trade name “CD117 MicroBead Kit”, “MS MACS (R) Columns”, “MiniMACS (TM) Separator”, all Miltenyi Biotech). Was recovered. Subsequently, the collected CD117-positive cells were further cultured using an expansion medium.

  1 (a) to (c) are optical micrographs of cultured amniotic fluid cells. FIG. 1A is a photograph at a magnification of 4 times, FIG. 1B is a photograph at a magnification of 10 times, and FIG. 1C is a photograph at a magnification of 20 times.

  Subsequently, in order to confirm the properties of CD117-positive amniotic fluid cells, surface antigen markers were confirmed by flow cytometry. 2A to 2H are graphs showing the results of flow cytometry analysis. As a result, CD29 (FIG. 2 (a)), CD44 (FIG. 2 (b)), CD73 (FIG. 2 (c)), CD90 (FIG. 2 (d)), CD105 (FIG. 2), which are mesenchymal stem cell markers. 2 (e)) was positive. Further, CD14 (FIG. 2 (f)), CD34 (FIG. 2 (g)), and CD45 (FIG. 2 (h)), which are markers for monocytes, hematopoietic stem cells and leukocytes, were negative. The above results indicate that CD117-positive amniotic fluid cells express mesenchymal stem cell markers.

  In addition, CD117-positive amniotic fluid cells were induced to differentiate into bone, cartilage and adipose tissue, and it was examined whether or not they had the ability to differentiate into these tissues. FIG. 3 (a) is a photograph showing the results of confirming that CD117-positive amniotic fluid cells were induced to differentiate into bone and differentiated into bone by Alizarin Red S staining. The magnification is 20 times. FIG. 3 (b) is a photograph showing the result of confirming that CD117-positive amniotic fluid cells were induced to differentiate into adipose tissue and differentiated into fat by Oil Red staining. The magnification is 20 times. FIG. 3 (c) is a photograph showing the results of confirming that CD117-positive amniotic fluid cells were induced to differentiate into cartilage tissue and differentiated into cartilage tissue by Alcian Blue staining and hematoxylin-eosin (HE) staining. The magnification is 40 times. As a result, it was confirmed that CD117-positive amniotic fluid cells have the ability to differentiate into bone, cartilage and adipose tissue.

  From the above results, it was confirmed that CD117-positive amniotic fluid cells can be used as mesenchymal stem cells.

[Experiment 3]
(Manufacture of cell sheets)
When a temperature-responsive polymer is used, cells can be detached from the dish by changing the temperature without performing trypsin treatment or the like. Therefore, an attempt was made to produce a cell sheet with CD117-positive amniotic fluid cells by using a dish coated with a temperature-responsive polymer.

  As the dish, a dish (trade name “Up Cell (registered trademark) 3.5 cm dish”, Cellseed) in which poly-N-isopropylacrylamide (PIPAAm), which is a temperature-responsive polymer, was uniformly immobilized was used. . In this dish, the surface of the substrate reversibly changes to hydrophobic and hydrophilic at 32 ° C.

  As a result of examining the cell density, culture time, and the like to be seeded on the dish, a cell sheet was successfully produced with CD117-positive amniotic fluid cells under the following conditions.

First, CD117-positive amniotic fluid cells are seeded in the above dish (trade name “Up Cell (registered trademark) 3.5 cm dish”, Cellseed)) at 3 to 5 × 10 ⁶ cells / dish and cultured at 37 ° C. for 3 days. did. As CD117-positive amniotic fluid cells, cells having 5 to 10 passages were used. Subsequently, by incubating the dish at 20 to 25 ° C. for about 15 minutes, physically intact cells were detached into a sheet shape, and the cell sheet was collected. FIG. 4 shows a representative photograph of the produced cell sheet.

As a result of examining the number of cells used for the production of the cell sheet, when cells are seeded at a cell density of 3 × 10 ⁶ cells / 3.5 cm dish (about 3 × 10 ⁵ cells / cm ² ) or more, sufficient strength for handling is obtained. There existed a tendency which can obtain the cell sheet of the thickness which has.

[Experimental Example 4]
(Analysis of cell sheet)
Paraffin-embedded sections of cell sheets prepared in the same manner as in Experimental Example 3 were prepared and examined for histopathology. Specifically, hematoxylin and eosin (HE) staining and immunostaining of vimentin, a mesenchymal cell marker, were performed.

  FIG. 5A is a photograph showing the result of HE staining of the cell sheet. FIG. 5 (b) is a photograph showing the result of staining a cell sheet with an anti-vimentin antibody. As a result, it was revealed that the cell sheet prepared with CD117-positive amniotic fluid cells is vimentin-positive and has the characteristics of mesenchymal cells from which amniotic fluid is derived.

[Experimental Example 5]
(Examination of the effect of cell sheet on mouse skin wound healing)
Inhalation anesthesia with isoflurane was introduced into 8-week-old male BALB / c mice. Subsequently, two full-thickness skin wounds were prepared on the back of the mouse. One of the wounds was the treatment group and the other was the control group. In the treatment group, a cell sheet (3 × 10 ⁶ cells) prepared in the same manner as in Experimental Example 3 was attached to the wound. On the other hand, nothing was affixed to the control group. Subsequently, wounds were observed on days 7, 14, and 21 after wound preparation.

  FIG. 6 (a) is a representative photograph of the wound of the control group. Moreover, FIG.6 (b) is a typical photograph of the wound part of a treatment group. FIG. 7 is a graph showing changes in the wound area of the control group and the treatment group. In FIG. 7, the wound area is shown in a ratio where the area of the wound produced is 100%.

  As a result, there was no significant difference in the reduction rate of the wound area between the control group and the treatment group. From this result, it was clarified that the wound was healed without causing a rejection reaction and delaying wound healing despite transplantation of a cell sheet derived from human cells into a non-immune-deficient mouse.

  In addition, mice were euthanized by cervical dislocation 14 and 21 days after wound preparation, and then skin tissue was collected and fixed with 4% paraformaldehyde phosphate buffer to prepare paraffin-embedded sections.

  Subsequently, the prepared tissue sections were stained with various staining methods (HE staining, Masson trichrome staining, Elastica van Gieson staining, Picrosirius red staining), and histological evaluation was performed. The formation and collagen composition were compared.

  Masson trichrome staining is a staining method that separates collagen fibers from muscle fibers by selectively staining collagen fibers. Elastica One-Geeson staining is a simultaneous multi-purpose staining method in which elastic fibers and collagen fibers in connective tissues are dyed separately and can be differentiated from muscle fibers. Picrosirius red staining is a staining method that separates type I collagen and type III collagen fibers in a tissue section.

  As a result, when the re-epithelialization rate of the wound on the 14th day after wound preparation was compared by Masson trichrome staining, no significant difference was observed between the two groups (n = 5). In addition, when the granulation tissue area on the 14th day after wound preparation was evaluated by elastica-van Gieson staining, no significant difference was observed between the two groups (n = 5).

  On the other hand, on the 21st day after wound preparation, the wound was healed in both the control group and the treatment group. However, the tissue of the wound on the 21st day after wound preparation was stained with Picrosirius red, and the collagen composition was determined by observation with a polarizing microscope. When examined, overexpression of type I collagen was observed in the control group, whereas in the treatment group, expression of type I collagen was low, and a large proportion of type III collagen was observed (n = 5).

  8A and 8B are photographs showing the results of Picrosirius red staining. FIG. 8 (a) shows the results of the treatment group, and FIG. 8 (b) shows the results of the control group. Overexpression of type I collagen during the wound healing process is a phenomenon that causes keloidization and scarring of the wound. From this result, by applying this cell sheet to the affected area, it is possible to heal wounds without delay without causing rejection, and it is possible to suppress keloidization and wound scarring which are complications of wounds. It became clear.

  ADVANTAGE OF THE INVENTION According to this invention, the technique which applies amniotic fluid stem cells to a surgical treatment and various skin care can be provided.

Claims (7)

  A cell sheet containing amniotic fluid stem cells.   The cell sheet according to claim 1, which is used for autologous transplantation or allogeneic transplantation.   The cell sheet according to claim 1 or 2, wherein the amniotic fluid stem cells are CD117 positive cells. The cell sheet according to claim 1, wherein the cell density is 3 × 10 ⁵ cells / cm ² or more. Recovering CD117 positive amniotic fluid cells;
Inoculating the collected cells on a culture substrate at a density of 3 × 10 ⁵ cells / cm ² or more and culturing in a sheet form;
A step of peeling and collecting the cells in a sheet form from the culture substrate;
A method for producing a cell sheet, comprising:   The manufacturing method according to claim 5, wherein the culture substrate is a culture substrate having a surface coated with a temperature-responsive polymer.   A cell sheet produced by the production method according to claim 5 or 6.