JP2019136019A - Manufacturing method of human fat tissue-derived somatic stem cells - Google Patents

Abstract

To obtain a large amount of human fat tissue-derived somatic stem cells holding high differentiation potency to an extent where the cells can be practically used for regenerative medicine.SOLUTION: There is provided a manufacturing method of human fat tissue-derived somatic stem cells in which human fat tissue-derived cells attached to a solid phase are separated into cells which are easily detached from the solid phase and cells which are difficult to be detached from the solid phase, the separated easily detachable cells are cultured in low oxygen under 7% of oxygen concentration, by which a large amount of human fat tissue-derived somatic stem cells holding high differentiation potency can be obtained to an extent where the cells can be practically used for regenerative medicine.

Description

The present invention relates to a method for producing human adipose tissue-derived somatic stem cells.

As stem cells used for regenerative medicine, there are somatic stem cells. Somatic stem cells are collected from the living body and are considered to be highly safe.

Somatic stem cells can be collected from various sites in the body and collected from adipose tissue. Somatic stem cells collected from human adipose tissue are called human adipose tissue-derived somatic stem cells. Unlike somatic stem cells derived from other tissues, human adipose tissue-derived somatic stem cells are said to have the ability to differentiate into three germ layers consisting of endoderm, mesoderm, and outer lung lobe, and are expected to be highly versatile. Is done. Conventionally, human adipose tissue-derived somatic stem cells have been prepared by various methods. However, in the conventional method, even if human adipose tissue-derived somatic stem cells are cultured, aging occurs before the sufficient amount is obtained, proliferation stops, and high differentiation ability is not maintained. It was considered difficult to put stem cells into regenerative medicine.

Hypoxic culture is known as a condition for artificially culturing cells. Hypoxic culture is said to mimic physiological oxygen conditions in vivo, and it is said that differentiation and proliferation ability of stem cells are improved by hypoxic culture. However, in these prior arts, the influence of the hypoxic culture on the differentiation ability or the proliferation ability was about several times enhancement and weak. In the case of human adipose tissue-derived somatic stem cells, practical application to regenerative medicine is difficult even if the number of cells is several times the conventional number.

An object of the present invention is to obtain a large amount of human adipose tissue-derived somatic stem cells that retain high differentiation potential to a practical level for regenerative medicine.

The present inventors, among human adipose tissue-derived cells adhering to the solid phase, maintain a high differentiation potential for a very long time under a hypoxic condition. In order to continue to proliferate efficiently without aging, it has been found that by including such proliferation in the production process, a large amount of cells that can be used in regenerative medicine can be obtained. In the case of the conventional method used without separating the cells adhering to the solid phase into cells that are easy to peel and cells that are difficult to peel, such a remarkable result is not observed even when cultured under low oxygen conditions. Therefore, this finding is completely unexpected. Hypoxic conditions are presumed to have a specific effect on cells that are easily detached.

That is, the present invention relates to a method for producing human adipose tissue-derived somatic stem cells, wherein human adipose tissue-derived cells attached to the solid phase are divided into cells that are easily detached from the solid phase and cells that are difficult to separate from the solid phase. Provided is a production method characterized by including a step of culturing the separated and easily detached cells under low oxygen.

In addition, the present invention provides the above production method, wherein the conditions for hypoxic culture are set by using as an index that the expression level of a hypoxia-inducing factor in cells does not exceed a predetermined value.

In addition, the present invention provides a method for separating cells that are easily detached from the solid phase and cells that are difficult to separate from the solid phase by adding a cell remover to the solid phase to which cells derived from human adipose tissue are adhered. The production method described above is performed by partially exfoliating and obtaining exfoliated cells.

Moreover, this invention provides the human fat origin somatic stem cell manufactured by said manufacturing method.

In the production method of the present invention, cells derived from human adipose tissue attached to a solid phase are used.

Cells derived from human adipose tissue attached to the solid phase are obtained by adding cells derived from human adipose tissue to the solid phase and allowing them to grow and adhere to the solid phase. Cells derived from human adipose tissue to be added to the solid phase are obtained by dispersing the adipose tissue in a liquid, centrifuging, and collecting with a pellet or the like. The liquid in which the adipose tissue is dispersed contains a proteolytic enzyme such as exogenous collagenase. Adipose tissue is derived from mammals such as humans and mice. The step of obtaining cells derived from human adipose tissue from adipose tissue includes a step of removing red blood cells. The step of removing red blood cells uses density gradient centrifugation or specific gravity. The solid phase to which the cells derived from human adipose tissue are attached may be a culture vessel. Growth after adding human adipose tissue-derived cells to the solid phase is performed in a low glucose medium in the presence of carbon dioxide and under normal oxygen concentration conditions. The glucose concentration in the low glucose medium is, for example, 1 to 5 g / L. The concentration of carbon dioxide during growth is, for example, 3 to 10%. The oxygen concentration during growth is, for example, 15 to 30%. After cells derived from human adipose tissue are attached to the solid phase, the solid phase is washed to remove cells that did not adhere.

In the production method of the present invention, cells derived from human adipose tissue adhering to the solid phase are separated into cells that are easily detached from the solid phase and cells that are difficult to separate from the solid phase, and separated cells that are easily detached are used. .

In order to separate cells that are easy to detach from the solid phase and cells that are difficult to detach from the solid phase and obtain separated cells that are easy to detach, the cell detachment agent is applied to the solid phase to which cells derived from human adipose tissue are attached. May be added to partially exfoliate the attached cells to obtain exfoliated cells. The cell peeling agent to be added is a drug for peeling cultured cells from the container, and may be a solution containing ethylenediaminetetraacetic acid. The solution containing ethylenediaminetetraacetic acid may be a solution containing only ethylenediaminetetraacetic acid as a solute. The concentration of ethylenediaminetetraacetic acid in the solution is, for example, 0.1 to 1.5 g / L. Acquisition of the cells detached after the addition of the cell detachment agent is performed by obtaining cells suspended from the solid phase after the addition of the cell detachment agent.

In the production method of the present invention, the separated cells that are easily detached are cultured in low oxygen.

The hypoxic culture of the separated cells is performed by culturing the separated cells in a hypoxic culture at a cell density of 5000 to 25000 cells / cm 2 in a container containing a medium. The container used for culture may be coated with a glycoprotein such as fibronectin. Fibronectin is a cell adhesion molecule and a biological material that forms an extracellular matrix. The medium used for the culture contains a cell growth promoter containing peptide hormones, a synthetic corticosteroid, an antioxidant, a growth factor, serum and the like. The concentration of oxygen in the low oxygen culture is, for example, about 7%. Hypoxic culture of cells is performed in the presence of carbon dioxide. The concentration of carbon dioxide is, for example, 4 to 10%. The culture temperature in the low oxygen culture is, for example, 33 to 39 ° C.

It is known that the expression level of a hypoxia inducing factor tends to increase under hypoxic conditions. However, it has been clarified that the production method of the present invention achieves a higher degree of proliferation under hypoxic conditions that do not increase the expression level of hypoxia-inducing factor in cells. Therefore, it is preferable to set the conditions for the hypoxic culture in the production method of the present invention as an index that the expression level of the hypoxia-inducing factor in the cells does not exceed a predetermined value. Thereby, a large amount of cells can be obtained more reliably. The culture conditions to be set are, for example, culture time and oxygen concentration during culture.

A hypoxia-inducing factor is a protein that is induced when oxygen supply to a cell falls short, and is a factor that functions as a transcription factor.

The amount of hypoxia-inducible factor expressed in the cell is measured, for example, by isolating a part of the cells in the hypoxic culture and detecting the hypoxia-inducible factor using a western blotting method or the like on the separated cells. be able to. The hypoxia inducing factor to be measured is preferably a hypoxia inducing factor protein. The expression level is measured based on the label attached to the hypoxia-inducing factor.

The predetermined value, which is an index for setting the hypoxic culture conditions, may be appropriately set according to the experimental system for measuring the expression level, the purpose of acquiring the cells, and the like. For example, the predetermined value is set with reference to the expression level under normal oxygen conditions (for example, 25%) or the expression level under low oxygen culture conditions of 2% or less.

The conditions for hypoxic culture may be adjusted over time based on the expression level of the hypoxia-inducible factor measured over time during the cell growth process. In this case, conditions can be set more delicately, whereby a larger amount of cells can be obtained.

According to the present invention, human adipose tissue-derived somatic stem cells that retain high differentiation ability can be obtained in a large amount that can be used in regenerative medicine.

Claims (4)

A method for producing human adipose tissue-derived somatic stem cells, wherein human adipose tissue-derived cells attached to the solid phase in the production process are separated into cells that are easily detached from the solid phase and cells that are difficult to separate from the solid phase, Characterized in that it comprises a step of low-oxygen culture of separated cells that are easily detached, and the conditions for the low-oxygen culture are set with an indication that the expression level of the hypoxia-inducing factor in the cells does not exceed a predetermined value. Production method. The production method according to claim 1, wherein the oxygen concentration is set to about 7% under conditions of low oxygen culture of human adipose tissue-derived somatic stem cells. A method for producing human adipose tissue-derived somatic stem cells, in which human adipose tissue-derived cells attached to the solid phase are separated into cells that are easily detached from the solid phase and cells that are difficult to separate from the solid phase, and separated. A production method comprising a step of culturing low-oxygen cells under an oxygen concentration of about 7% of cells that are easily treated. Separation of cells that are easy to detach from the solid phase and cells that are difficult to detach from the solid phase is performed by adding a cell detaching agent to the solid phase to which cells derived from human adipose tissue are attached, and partially detaching the attached cells. The manufacturing method of any one of Claims 1-3 performed by acquiring the peeled cell.