KR100902569B1 - Pericyte Derived From Human Umbilical Cord And Method For Establishing the Same - Google Patents

Abstract

The present invention relates to human umbilical cord-derived mesenchymal stem cells and a method for establishing the same. More particularly, the present invention relates to a method for separating mesenchymal stem cells from human umbilical cord with high purity. The present invention relates to a method for culturing the isolated human cord-derived mesenchymal stem cells and a method for freezing and thawing human cord-derived mesenchymal stem cells. The present invention also relates to a culture medium for culturing mesenchymal stem cells isolated from human umbilical cord. The present invention also relates to human umbilical cord-derived mesenchymal stem cells isolated, cultured, frozen and / or thawed by the above method.

The separation method of human umbilical cord-derived mesenchymal stem cells of the present invention can be separated / purified with high purity by a simple method, and furthermore, the human umbilical cord-derived mesenchymal stem cells of the present invention are characterized by having high viability and proliferative capacity. .

Umbilical cord, mesenchymal stem cells derived from human umbilical cord, mesenchymal stem cells, α-SMA

Description

Pericyte Derived From Human Umbilical Cord And Method For Establishing the Same}

Figure 1 shows a cell picture of human umbilical cord-derived mesenchymal stem cells from passage 0 to passage 12, the vertical axis represents the passage and the horizontal axis is 100 times (X100), 200 times (X200), 400 times (X400) It is a photograph seen at magnification. Spindle-shape similar to fibroblasts were maintained from the beginning of the passage to late stages. (A) was 40-fold, (B) 200-fold, (C) 200-fold and (D) 400-fold. It is a photograph seen at a magnification of.

Figure 2 is a diagram confirming that the cells in the human umbilical cord-derived mesenchymal stem cells of the present invention forms a cell layer (heaping) rather than a monolayer (monolayer).

Figure 3 is a photograph showing the growth curve of human umbilical cord-derived mesenchymal stem cells of the present invention.

Figure 4 is a photograph showing the results of FACS (fluorescence activated cell sorting) analysis of human umbilical cord-derived mesenchymal stem cells of the present invention.

Figure 5 is a diagram showing the immunochemical staining results of human umbilical cord-derived mesenchymal stem cells of the present invention.

Figure 6 is a diagram showing the results of immunofluorescence staining of human umbilical cord-derived mesenchymal stem cells of the present invention.

The present invention relates to mesenchymal stem cells from the human umbilical cord and a method of establishing the same. More specifically, the present invention relates to a method for separating mesenchymal stem cells with high purity from human umbilical cord. The present invention also relates to a method for culturing the isolated human cord-derived mesenchymal stem cells and a method for freezing and thawing human cord-derived mesenchymal stem cells.

In another aspect, the present invention relates to a culture medium for culturing mesenchymal stem cells isolated from human umbilical cord.

In another aspect, the present invention relates to human umbilical cord-derived mesenchymal stem cells isolated, cultured, frozen and / or thawed by the above method.

Endothelial cells are present in the entire vascular system from the heart to capillaries and regulate the movement of oxygen and nutrients. Developmentally, arteries and veins develop in small blood vessels consisting only of small vascular endothelial cells and later follow signals from vascular endothelial cells to vascular mesenchymal stem cells, connective tissue, and smooth muscle cells. Muscle cells combine to form blood vessels. When blood vessels mature, they play an important role in transmitting signals from vascular endothelial cells to the surrounding cells and maintaining their function. Similarly, the surrounding cells also transmit signals to vascular endothelial cells.

 Vascular mesenchymal stem cells (pericyte) are vascular mural cells buried in the basement membrane of the microvascular system, which forms a specific local contact with the vascular endothelium. Although the presence and role of vascular mesenchymal stem cells have been underestimated for a long time, in recent years, perivascular cells have been attracting attention as essential elements for the formation of microvascular system, and are important regulators of blood vessel development, stabilization, maturation and remodeling. It's coming up.

 In general, vascular mesenchymal stem cells (pericyte) and vascular smooth muscle cells (VSMC) are regarded as the same cell lineage, but are distinguished by markers, shape, and location in contact with the vascular endothelium. However, this distinction of vascular mesenchymal stem cells from vascular myofibroblasts cannot be an absolute criterion. The most reliable method for identifying vascular mesenchymal stem cells is to determine whether the vascular endothelial cells and the basement membrane are shared by electron microscopy.

 The typical vascular myopic muscle cells in the aorta or vena cava are separated by the basement membrane, but the vascular mesenchymal stem cells are inside the vascular endothelial basement membrane. Vascular mesenchymal stem cells are interposed between vascular endothelial cells and the basement membrane, but are connected by a peg-socket junctional complex in the absence of the basement membrane. This is where intracellular substances can move. As a result, vascular mesenchymal stem cells and vascular endothelial cells are very closely related, and material exchange can be freely performed.

 These vascular mesenchymal stem cells (pericyte) is different from the vascular endothelial cells depending on the species (species) or (organ). Vascular endothelial cells and vascular mesenchymal stem cells are present at 1: 100 in skeletal muscle, 1:10 in lung, and 1: 1 in retina.

 Vascular mesenchymal stem cells are reported to be formed from smooth muscle cells, fibroblasts, endothelial cells, or bone marrow. Vascular mesenchymal stem cells also have the potential to differentiate into smooth muscle cells, fibroblasts, osteoblasts, chondrocytes, or adipocytes.

Vascular mesenchymal stem cells have been reported to block their function in intravascular tumors, which can prevent neovascularization of tumors. The study of perivascular cells is important for chemotherapy targeting tumor vascular mesenchymal stem cells. Vascular mesenchymal stem cells are also important for cerebral blood and barrier formation. The astrocytes and the brain capillary endothelial cells are anatomically close together. This suggests that mutual cooperation between the two can contribute to the formation of cerebrovascular barriers. However, culturing astrocytes and vascular mesenchymal stem cells in vitro does not perform as satisfactorily as compared with in vivo cerebrovascular barriers. Recent results suggest that there may be other important factors in addition to microvascular endothelial cells and astrocytes, and that vascular mesenchymal stem cells are involved in this function.

It is also reported to be important in the retina. Retinal capillaries consist of three basic structures: vascular endothelial cells, basement membrane, and vascular mesenchymal stem cells. In humans and rats, vascular endothelial cells and vascular mesenchymal stem cells are present in a ratio of almost 1: 1. The main features of diabetic retinopathy are vascular permeability and vascular occlusion. The role of vascular mesenchymal stem cells in this process is not clear. The basic shape change in the diabetic retina is the loss of vascular mesenchymal stem cells that produce capillaries that do not contain cells. The function of vascular mesenchymal stem cells to defend vascular endothelial cells in the hyperglycemic state is replaced by the repair of capillary endothelial cells. Two possibilities have recently been proposed for this phenomenon: toxic substances and cytotoxic signals generated in perivascular cells. The other is the removal of vascular mesenchymal stem cells by an active process.

 Vascular mesenchymal stem cells play an important role in various diseases and research models. Therefore, in order to study vascular mesenchymal stem cells, it is necessary to isolate and culture purely.

 Therefore, the inventor of the present invention isolates human umbilical cord-derived mesenchymal stem cells from human umbilical cord with simple and high purity, and succeeds in freezing and thawing for cultivation and storage thereof, thus leading to the present invention.

Therefore, the present invention is to provide a method for establishing mesenchymal stem cells derived from human umbilical cord. More specifically, the present invention is to provide a method for separating mesenchymal stem cells with high purity from human umbilical cord. In addition, the present invention is to provide a method for culturing the isolated human umbilical cord-derived mesenchymal stem cells and a method for freezing and thawing human umbilical cord-derived mesenchymal stem cells.

The present invention also provides a culture medium and its composition for culturing mesenchymal stem cells isolated from human umbilical cord.

On the other hand, the present invention is to provide human umbilical cord-derived mesenchymal stem cells isolated, cultured, frozen and / or thawed by the above method.

In order to accomplish the above technical problem, the human umbilical cord-derived mesenchymal stem cell establishment method of the present invention comprises the following steps.

That is, the method of establishing human umbilical cord-derived mesenchymal stem cells of the present invention comprises the steps of: (a) separating the artery from the umbilical cord; (b) incubating the isolated artery with collagenase, pronase and dienase for a period of time and then separating the suspended supernatant; And (c) separating and purifying human umbilical cord stem cells from the obtained cell mass of the suspended supernatant.

Compared with the prior art, after separating the artery and the umbilical cord from the umbilical cord of the present invention, the mesenchymal stem cells can be easily and efficiently obtained using a few enzymes to obtain human umbilical cord-derived mesenchymal stem cell masses.

In particular, in order to separate human umbilical cord-derived mesenchymal stem cells with high purity and high viability, it is necessary to adjust the culture time and temperature appropriately. After incubation with the enzyme, it is necessary to add up to 50 ml of HBSS to efficiently collect the cell mass.

In addition, in order to determine the purity of the isolated and cultured human umbilical cord mesenchymal stem cells should be confirmed using a specific marker (marker). Such markers are summarized in Table 4 described later.

Therefore, in order to effectively separate the artery from the human umbilical cord of step (a) and the umbilical cord from which the artery has been removed, giving a house with a scissor to Wharton's jelly outside the artery, in step (b) Incubation of the isolated arteries with collagenase, pronase and dienase comprises incubators for 2, 4, 6 hours in the range of 35-38 ° C., preferably at about 37 ° C. for 4 hours. In this case, the culture medium is not limited thereto, but 0.05% collagenase, 0.02% pronase, and 0.02% dienase may be used in a total amount of 5 to 10 ml.

In addition, in order to efficiently collect cell masses from the supernatant in step (c), prior to step (c), put 50 ml of HBSS in the supernatant supernatant, centrifuged and suspended in the culture medium to dish Sowing to a; further comprises.

In this case, the culture medium is not limited thereto, but DMEM and FBS may be used in a volume ratio of about 4: 1.

In addition, the step (c) further comprises the step of removing impurities by passing the cell mass through a mesh of 10 to 100㎛ range, more preferably about 30㎛. The use of a mesh in the present invention prevents contamination of other cells such as fibroblasts of connective tissue and reduces competition with other cells, thereby increasing viability in the dish. In particular, by using this method, human umbilical cord-derived mesenchymal stem cells can be isolated / purified with a purity of 90% or more.

On the other hand, the present invention relates to a method for establishing a human umbilical cord-derived mesenchymal stem cell line by subcultured human umbilical cord-derived mesenchymal stem cells obtained according to the above method. More specifically, the subculture is carried out at 60 to 80% confluence.

More specifically, the subculture includes (a) completely removing remaining FBS by washing with DMEM after removing the medium; (b) adding 0.05% trypsin / EDTA warmed for 5 to 15 minutes in a 35 to 38 ° C. water bath and storing in a CO ₂ incubator for 0.5 to 1.5 minutes; (c) lightly impacting the culture dish so that the cells are physically separated and then inactivated by injecting the same amount of medium with trypsin / EDTA to kick off the cells; And (d) centrifuging the cells at low temperature to remove supernatant and resuspend in medium to transfer to another culture dish; It includes.

In addition, the centrifugation of the step (d) includes the one made in a temperature range of 3 to 5 ℃, more preferably 4 ℃. In addition, the step (d) may further include the step of monitoring the number of cells or cell viability by staining the cell mass after removing the supernatant after centrifugation.

On the other hand, the present invention relates to mesenchymal stem cells and stem cell lines derived from the human umbilical cord obtained according to the above method. In particular, the human umbilical cord-derived mesenchymal stem cell line of the present invention is characterized by prominent expression of CD29, CD44, CD90, α-Smooth muscle actin (α-SMA) and NG2.

On the other hand, the present invention relates to a method of preserving human umbilical cord-derived mesenchymal stem cells and stem cell lines by freezing, specifically, (a) after preparing 1 x 10 ⁶ to 2 x 10 ⁶ cells and suspending the freezing medium Placing in a freezing vial and placing in a freezing box being stored at room temperature; And (b) after storing in the refrigeration box for 20 to 30 hours at -75 to -85 ° C transferred to a liquid nitrogen (LN ₂ ) tank for storage; It includes. In addition, the freezing medium is not limited thereto, but DMSO, FBS, and DMEM may be included in a volume ratio of about 5:10:35.

On the other hand, the present invention relates to a method for thawing frozen human umbilical cord-derived mesenchymal stem cells and stem cell lines according to the above method, specifically, (a) rapidly dissolving frozen vials in the temperature range of 35 to 38 ℃ Transferring to clean-bench before completely melting; (b) dropping dropwise using a pipette on a medium and centrifuging at a temperature ranging from 2 to 5 ° C .; And (c) suspending in dish by suspending in medium; It includes. At this time, the medium is not limited to this, FBS and DMEM is characterized in that it contains a volume ratio of 2: 8.

Hereinafter, the present invention will be described in detail according to specific embodiments. However, it is obvious that the scope of the present invention is not limited by the following examples.

<Example>

Example 1 Isolation / Identification and Freezing / Thawing of Human Umbilical Cord-derived Mesenchymal Stem Cells

1. Umbilical Cord Acquisition Process

10-20 cm of umbilical cord is completely submerged in a solution mixed with HBSS (Hanks' Balanced Salt Solution, JBI, 003-02) with 3 × Antibiotics (Gibco, 5240-062), gentamycin, plasmocin (Invivogen, ant-mpt) Transported at room temperature.

2. Isolation of Cells from the Umbilical Cord

After cutting the umbilical cord to about 3 ~ 4 ㎝ and using a scissor (scissor) to make an incision along the outer portion of the artery and tweezers to separate the artery and umbilical cord. 0.05% solution I (0.05% collagenase type I) (Gibco, 17100-017), 0.02% pronase (Roche, 165 921), 0.2% dienase (DNase, prestored at 37 ° C) I) Sigma was poured into a 50 ml tube about 5-10 ml and the artery was submerged.

Then, after storage for 2, 4, 6 hours in a 37 ℃ incubator (incubator) to recover the supernatant was passed through a 30㎛ mesh (mesh).

After filling up to 50ml HBSS was centrifuged for 10 minutes at 500g at 4 ℃. The supernatant was removed and then suspended again in 50 ml DMEM (JBI, L001-05) and centrifuged at 500 g for 10 minutes at 4 ° C. This process was repeated twice.

Finally, the supernatant was removed and then suspended in 1 ml culture medium (80 ml DMEM (JBI, L001-05) and 20 ml FBS (Hyclone, SH30088.03) (see Table 1). It was then seeded into 35 mm dishes.

3. Culture method

Subcultures were performed when 70% confluency was achieved after the first human umbilical cord-derived mesenchymal stem cell isolation / culture. The highest viability and proliferation were seen at about 70% confluency.

Cell images of passaged human umbilical cord-derived mesenchymal stem cells from passage 0 to passage 12 are summarized in FIG. 1. In Figure 1, the vertical axis represents the passage and the horizontal axis is a photograph viewed at a magnification of 100 times (X100), 200 times (X200), 400 times (X400), and subcultured spindle-shape similar to fibroblasts. Maintained from early to late. This shape was confirmed to be similar to mesenchymal stem cells derived from human bone marrow (bone marrow).

In addition, in the photographs seen at magnifications of 100 times (X100), 200 times (X200), and 400 times (X400) in FIG. 1, cells in the human umbilical cord-derived mesenchymal stem cells of the present invention are monolayer. In addition, it was confirmed that the cell layer (heaping) is also achieved, which is shown in FIG.

On the other hand, the composition of the culture medium (100 ml) for culturing human umbilical cord-derived mesenchymal stem cells is shown in Table 1 below.

ingredient volume DMEM 80 ml FBS 20 ml

Human umbilical cord-derived mesenchymal stem cell passage method of the present invention was performed as follows.

(1) After removing the medium, it was washed once with DMEM to completely remove the remaining FBS.

(2) 0.05% trypsin / EDTA, which was warmed for about 10 minutes in a water bath at 37 ° C., was placed in the CO ₂ incubator for 1 minute after the cells were submerged.

(3) The dish was lightly impacted to allow the cells to physically fall off. (4) Inactivated the cells after adding the same amount of trypsin / EDTA as the medium.

(5) Centrifuged at 500g for 5 minutes at 4 ° C, the supernatant was removed and suspended in media to examine cell number and viability using tryphan blue.

In addition, the number of cells sown and the size of dishes in the cell passage culture of the present invention preferably satisfies the values summarized in Table 2 below.

Dish size Number of cells sown 35 mm 0.5 x 10 ⁵ 60 mm 1 x 10 ⁵ 100 mm 3 x 10 ⁵

   Growth curves of human umbilical cord stem cells are shown in FIG. 3. As shown in FIG. 3, no senescence phenomenon was observed during the subculture and stable growth was observed.

4. Identification of High Performance / Purity Human Umbilical Cord-derived Mesenchymal Stem Cells

For high purity isolation of human umbilical cord-derived mesenchymal stem cells, it is necessary to adjust the culture time and temperature appropriately. In addition, in order to determine the purity of the cells isolated and cultured, specific markers expressed only in mesenchymal stem cells should be identified.

5 . FACS Analysis of Human Umbilical Cord-derived Mesenchymal Stem Cells

Markers for FACS (fluorescence activated cell sorting) analysis of human umbilical cord-derived mesenchymal stem cells are summarized in Table 3 below.

Marker Another name Expression CD29 β1 integrin Lymphocytes, smooth muscle cells, epithelial cells CD44 H-CAM Lymphocyte, Mesenchymal Stem Cells CD90 Thy-1 Hematopoietic stem cells, mesenchymal stem cells α-SMA α-smooth muscle actin Various muscle cells, mesenchymal cells and mesenchymal stem cells

FACS analysis of human umbilical cord-derived mesenchymal stem cells was performed as follows.

(1) ⁵ × 10 ⁵ or less cells were put in a FACS tube, 2 ml of FACS buffer was added, and centrifuged at 500 ° C. for 5 minutes at 4 ° C.

(2) In order to remove the supernatant and analyze the antigen in the cell, the suspension was suspended in 500 µl of PBS, and 1200 µl of 100% EtOH stored in ice was dropped one by one with vortexing.

(3) Incubated on ice for 30 minutes.

(4) 2 ml of FACS buffer was added and centrifuged for 5 min at 500 g at 4 ° C. The subsequent process is the same for intracellular and external antigen analysis.

(5) The cells were suspended in 100 µl supernatant.

(6) The fluorescent antibody was treated and incubated on ice again for 30 minutes.

(7) 2 ml of FACS buffer was added and centrifuged for 5 min at 500 g at 4 ° C.

(8) The cells were suspended in 100 µl supernatant.

(9) If the fluorescent secondary antibody is needed, repeat the above (6-7).

(10) Analyzed by FACSCalubur.

The FACS of human umbilical cord-derived mesenchymal stem cells of the present invention was analyzed and the results are summarized in FIG. 4.

As shown in Figure 4, the FACS analysis of human umbilical cord-derived mesenchymal stem cells CD29, CD44, CD90, alpha smooth muscle all showed more than 90% expression and composed of highly uniform human umbilical cord-derived mesenchymal stem cells I could see that.

6 . Immunohistochemical Analysis of Human Umbilical Cord-derived Mesenchymal Stem Cells

 Markers for immunohistochemistry analysis of human umbilical cord-derived mesenchymal stem cells are summarized in Table 4 below.

Marker Another name Expression NG2  NG2 Chondroitin Sulfate Proteoglycan Oligodendocyte α-SMA α-smooth muscle actin Various muscle cells, mesenchymal cells and mesenchymal stem cells Desmin - Expressed in various muscle cells

Immunohistochemical analysis of human umbilical cord-derived mesenchymal stem cells was performed as follows.

(1) Incubated in 50 ml methanol containing 700 µl of H ₂ O ₂ at room temperature for 30 minutes. (2) Washed three times on a shaker with PBS for 5 minutes.

(3) Incubated for 15 minutes in 0.5% Triton-X100.

(4) Washed three times on a vibrator for 5 minutes with PBS.

(5) After the water was removed, it was treated with 30 μl of normal goat serum at room temperature for 1 hour.

(6) After removal of normal goat serum, 30 μl of primary antibody was treated.

(7) incubated for 1 hour at room temperature.

(8) Washed three times on vibrator for 5 minutes with PBS.

(9) After removing water, 30 μl of secondary antibody was treated for 1 hour.

(10) Washed three times on vibrator for 5 minutes with PBS.

(11) Color development for 30 seconds to 1 minute and 30 seconds using DAB was followed by washing for 10 minutes in running tap water.

(12) Hematoxylin was used to stain the nuclei, followed by washing for 10 minutes in running tap water.

(13) After dehydration in the order of 70%, 85%, 95%, 100% EtOH, and incubated for 10 minutes in xylene (Xylene).

(14) The cover glass was mounted by mounting with a fixed solution and observed with an optical microscope.

The results are shown in FIGS. 5 and 6.

As shown in FIG. 5, it can be seen that most of the cells are expressed from the early stage to the late stage of NG2 and α-SMA in the marker. In addition, as shown in FIG. 6, it can be seen that NG2 and α-SMA are simultaneously expressed in most cells through immunofluorescence staining.

7. Human Umbilical Cord Origin Mesenchyme  Freezing and thawing stem cells

Human umbilical cord-derived mesenchymal stem cells isolated and identified according to the present invention were stored frozen by the following method.

(1) 1-2 × 10 ⁶ cells were prepared.

(2) After carefully suspending in 1 ml of freezing media, they were placed in freezing vials and placed in cryo-boxes stored at room temperature, and then stored at -80 ° C for one day.

(3) Transferred to LN ₂ tank and stored.

The preferred composition of the freezing medium (100 mL) used in the present invention is shown in Table 5 below.

Furtherance ingredient DMSO 5 ml FBS 10 ml DMEM 35 ml

In addition, the human umbilical cord-derived mesenchymal stem cells of the present invention stored by the above method was thawed by the following method and used as necessary.

(1) Frozen vials were rapidly dissolved at 37 ° C. and transferred to a clean-bench before being completely dissolved.

(2) Dropped dropwise by using a pipette on 9 ml of medium. (3) The mixture was centrifuged at 500 g for 5 minutes at 4 ° C.

(4) It was suspended in 1 ml medium and seeded in a dish.

On the other hand, the preferred composition of the freezing medium (100 mL) used in the present invention is shown in Table 6.

Furtherance ingredient FBS 20 ml DMEM 80 ml

The separation method of human umbilical cord-derived mesenchymal stem cells of the present invention can be isolated / purified with high purity by a simple method, and furthermore, the human umbilical cord-derived mesenchymal stem cells of the present invention can be cultured for a long time with high survival rate. It is done.

Claims (24)

(a) separating the artery from the umbilical cord; (b) incubating the isolated artery with collagenase, pronase and dienase in the range of 35 to 38 ° C. for 2 to 6 hours and then separating the floating supernatant; And (c) isolating and purifying human umbilical cord derived stem cells from the cell mass collected from the obtained supernatant supernatant; and obtaining human umbilical cord derived mesenchymal stem cells. The method of claim 1, wherein the artery in step (a) is isolated by dispensing a house with a scissor to the outer Whartons jelly. delete The method of claim 1, wherein the culture solution is characterized in that the use of 0.05% collagenase, 0.02% pronase and 0.02% dienase in a total amount of 5 to 10ml range. According to claim 1, prior to step (c), HBSS in the suspended supernatant to 50ml by centrifugation and then suspended in the culture medium sowing in a dish; characterized in that it further comprises . The method of claim 5, wherein the medium comprises DMEM and FBS in a volume ratio of 4: 1. The method of claim 5, wherein the dish uses a dish in the range of 35 to 100 mm. The method of claim 1, wherein the cell mass collected in the step (c) is further passed through a mesh in a range of 10 to 100 µm to remove impurities. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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