JP2010063404A - Method for separating stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating a stem cell in a healthy state from a biological tissue such as fat tissue, bone marrow, etc., or blood such as peripheral blood, etc., in a short time period. <P>SOLUTION: The method for separating the stem cell includes a step S3 for storing a cell suspension containing mononucleosis cells isolated from a biological tissue in a hydrophilic container and allowing the suspension to stand still so as to form spheroids and a step S4 for separating the formed spheroids by size. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for separating stem cells.

  2. Description of the Related Art Conventionally, a biological tissue processing apparatus that isolates adipose-derived cells from adipose tissue by washing adipose tissue collected from a human and stirring it with a digestive enzyme solution is known (for example, see Patent Document 1). Since the adipose-derived cells isolated by this biological tissue processing apparatus include other mononuclear cells in addition to stem cells, the isolated adipose-derived cells are included to extract only stem cells. By seeding and culturing a cell suspension in a petri dish that has been subjected to hydrophobic treatment, stem cells are adhered to the bottom of the petri dish and separated using the adhesiveness of the stem cells.

WO05 / 012480 pamphlet

  However, the conventional method of separating stem cells by adhering them to a petri dish has the disadvantage that it takes time to culture. In addition, in order to peel off the adhered stem cells, it is necessary to add a proteolytic enzyme such as trypsin, and there is a disadvantage that the stem cells are damaged. Furthermore, it has been reported that the properties of stem cells may change when they adhere to a petri dish.

  The present invention has been made in view of the above-described circumstances, and is capable of separating stem cells from living tissue such as adipose tissue and bone marrow or blood such as peripheral blood in a short time and in a healthy state. It aims to provide a method.

In order to achieve the above object, the present invention provides the following means.
The present invention comprises a step of forming a spheroid by storing a cell suspension containing mononuclear cells isolated from living tissue or blood in a hydrophilic container and allowing the spheroid to be sized. And a method for separating stem cells.

  According to the present invention, when a cell suspension containing mononuclear cells isolated from living tissue or blood is contained in a hydrophilic container, stem cells in the mononuclear cells also adhere to the bottom surface of the hydrophilic container. Can not be maintained and remains floating. Therefore, by standing in that state, stem cells having adhesiveness adhere to each other to form spheroids. Since spheroids are different in size from other mononuclear cells, they can be easily separated from other mononuclear cells in the separation step.

  That is, according to the present invention, stem cells can be extracted from mononuclear cells without performing culture treatment, and can be separated and recovered by proteolytic enzyme without peeling from the culture vessel. As a result, the time required to separate the stem cells can be shortened and the stem cells in a healthy state can be collected without causing damage.

In the above invention, the step of separating the spheroids may be performed by separating the spheroids from other mononuclear cells using a filter.
By doing in this way, the stem cell which comprised the spheroid is captured without passing through a filter, and can be easily separated from other mononuclear cells smaller than that passing through the filter.

Moreover, in the said invention, the step which isolate | separates the said spheroid is good also as separating a spheroid from another mononuclear cell cell by crossflow filtration.
By doing so, spheroids flow through the main channel and are collected, and mononuclear cells having a small size pass through the membrane and are easily separated.

  According to the present invention, there is an effect that stem cells can be separated from a living tissue such as adipose tissue and bone marrow or blood such as peripheral blood in a short time and in a healthy state.

A stem cell separation method according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the method for separating stem cells according to this embodiment comprises step S1 of isolating mononuclear cells from a living tissue, for example, adipose tissue, and erythrocytes from the isolated mononuclear cells. Step S2 for removing, Step S3 for forming spheroids from mononuclear cells from which red blood cells have been removed, and Step S4 for separating the formed spheroids from other mononuclear cells.

  Step S1 for isolating mononuclear cells is performed by mixing and stirring the adipose tissue and the digestive enzyme solution. A cell suspension is produced in which adipose tissue is degraded and mononuclear cells are isolated.

  In step S2 for removing red blood cells, the cell suspension generated in step S1 is centrifuged to separate red blood cells in mononuclear cells by the difference in specific gravity. By selectively removing the separated red blood cells, mononuclear cells that do not contain red blood cells can be obtained. Instead of centrifugation, red blood cells may be selectively removed by adding a low osmotic pressure solution to the cell suspension.

  Step S3 for forming spheroids is performed by seeding the mononuclear cells obtained in step S2 in a dish subjected to hydrophilic treatment (for example, 2-methacryloyloxyethylphosphorylcholine treatment) and leaving them to stand. Although any dish can be adopted, the formation of spheroids is promoted by adopting a U-bottom multi-well plate. Thereby, a spheroid is formed as shown in FIG.

  In step S4 for separating spheroids, the cell suspension containing the spheroids formed in step S3 is allowed to pass through a porous filter having pores smaller in diameter than spheroids and larger than single monocyte cells alone. As a result, the spheroids are trapped by the filter and remain, and other mononuclear cells smaller in size pass through the through holes without being captured by the filter and are separated from the spheroids. And spheroids are collect | recovered by taking out the spheroids captured by the filter from the filter by backwashing or the like (see FIG. 3).

  As a result of seeding the separated and recovered spheroids and culturing them in a differentiation-inducing medium, differentiation into fat (see FIG. 4) and bone differentiation (see FIG. 5) was confirmed, and the spheroids are stem cells having multipotency. It was confirmed that there was.

  Thus, according to the cell separation method according to the present embodiment, unlike the conventional method of recovering cells adhering to the bottom of the dish as stem cells by culturing the cell suspension in a dish, the culture is performed. Stem cells can be isolated without performing Therefore, the time conventionally required for culture can be saved, and stem cells can be collected within a short time.

  In addition, the conventional method for separating stem cells by culturing does not involve detaching the stem cells adhered to the dish by culturing, so that the stem cells collected by proteolytic enzymes can be prevented from being damaged, and healthy stem cells can be removed. It can be recovered.

  In the present embodiment, the spheroids are separated from other mononuclear cells by a filter, but instead, as shown in FIG. 6, a cross flow filtration filter (for example, seiler company) It is good also as separating by 1).

  The cross flow filtration filter 1 is smaller than the spheroid A and larger than the mononuclear cell B (for example, the diameter of 10 μm to 50 μm, preferably 50 μm to 100 μm). A cross flow membrane 4 is arranged on the inner surface, and a cell suspension C containing spheroid A and other mononuclear cells B is caused to flow in the main flow path 3. Thereby, the other mononuclear cells B are discharged from the main flow path 3 through the cross flow membrane 4 and the porous body 2, and the spheroid A can be recovered at the outlet of the main flow path 3.

It is a flowchart which shows the isolation | separation method of the stem cell which concerns on one Embodiment of this invention. It is a microscope picture which shows the state in which the spheroid is formed in step S3 of the separation method of FIG. It is a microscope picture which shows the spheroid isolate | separated in step S4 of the isolation | separation method of FIG. It is a microscope picture which shows the fat obtained as a result of culturing the spheroid isolate | separated by the isolation | separation method of FIG. 1 using the differentiation-inducing medium. It is a microscope picture which shows the bone obtained as a result of having culture | cultivated the spheroid isolate | separated by the isolation | separation method of FIG. 1 using the differentiation-inducing medium. It is a figure which shows the modification of the filter used for step S4 of the separation method of FIG.

Explanation of symbols

S3 Step of forming spheroids S4 Step of separating spheroids 1 Crossflow filtration filter (filter)

Claims (3)

Forming a spheroid by containing a cell suspension containing mononuclear cells isolated from biological tissue or blood in a hydrophilic container and allowing to stand;
Separating the formed spheroids according to size.   The method for separating stem cells according to claim 1, wherein the step of separating the spheroids separates the spheroids from other mononuclear cells using a filter.   The method for separating stem cells according to claim 2, wherein the step of separating the spheroids separates spheroids from other mononuclear cells by cross-flow filtration.

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