HK1180545B - Cryopreservation of umbilical cord tissue for cord tissue-derived stem cells - Google Patents

Abstract

A method of preserving an umbilical cord is disclosed. The method comprises obtaining a segment of an umbilical cord; mincing the segment of the umbilical cord into cord tissue pieces; admixing the cord tissue pieces with a cryogenic composition comprising a cryoprotectant and a protein to form a mixture; shaking the mixture for a duration of no shorter than 20 minutes and no longer than 40 minutes; and cryopreserving the mixture.

Description

Umbilical cord tissue cryopreservation suitable for umbilical cord tissue-derived stem cells

Technical Field

The present invention relates to stem cells, and in particular to umbilical cord tissue-derived stem cells.

The priority of taiwan patent application No. 100131519, filed on 9/1/2011, the entire contents of which are hereby incorporated by reference.

Background

Previously suggested potential benefits of preserving infant cord blood, scientists now further preserved the actual cord tissue. The idea is to preserve a section of umbilical cord at birth and all the cells within it and to freeze the umbilical cord tissue in a cryogenic reservoir to facilitate long term preservation. In the future, if cells from the infant are needed for treatment, the umbilical cord tissue can be treated by the most advanced techniques at that time to extract the cells.

Wharton's jelly in the umbilical cord (umbilical cord tissue) is rich in multipotent Mesenchymal Stem Cells (MSC), which has great potential in regenerative medicine. MSCs can be differentiated into skeletal, cartilage, neural, adipose, cardiac, smooth muscle, liver and skin cells.

However, the above-mentioned methods are quite novel in terms of preservation and subsequent cell culture, and an efficient process is still needed.

Disclosure of Invention

In one embodiment, the present invention is directed to a method of preserving an umbilical cord. The method comprises the following steps: obtaining a section of umbilical cord; mincing an umbilical cord into umbilical cord tissue pieces, wherein each umbilical cord tissue piece is no greater than 2 millimeters (mm) in size; mixing the umbilical cord tissue pieces with a freezing composition to form a mixture, wherein the freezing composition comprises a cryoprotectant and a protein; shaking the mixture for a period of not less than 28 minutes but not more than 32 minutes; the mixture was then stored frozen.

In another embodiment, the invention is directed to a method of preserving an umbilical cord comprising: obtaining a section of umbilical cord; mincing the umbilical cord into umbilical cord tissue pieces; mixing the umbilical cord tissue pieces with a freezing composition to form a mixture, wherein the freezing composition comprises a cryoprotectant and a protein; shaking the mixture for a period of not less than 20 minutes but not more than 40 minutes; the mixture was then stored frozen.

In another embodiment, the invention is directed to a method of obtaining umbilical cord tissue-derived stem cells from a section of umbilical cord comprising: obtaining a section of umbilical cord; mincing the umbilical cord into umbilical cord tissue pieces; mixing the umbilical cord tissue pieces with a freezing composition to form a mixture, wherein the freezing composition comprises a cryoprotectant and a protein; shaking the mixture for a period of not less than 20 minutes but not more than 40 minutes; freezing and preserving the mixture; thawing the mixture and removing the frozen composition; the umbilical cord tissue pieces are then cultured in a culture medium to obtain umbilical cord-derived stem cells.

In another embodiment, the present invention is directed to a frozen composition comprising: a) serum albumin or human serum; and b) a cryoprotectant, wherein the composition is free of non-human animal derived components.

In another embodiment, the invention relates to a frozen composition comprising 30% to 50% human umbilical cord blood serum or 2.5% to 25% serum albumin; 5.5% to 55% dimethyl sulfoxide (DMSO); and 0.5% to 5% dextran.

The foregoing and other embodiments will be apparent from the following description of the preferred embodiments, taken in conjunction with the accompanying drawings. Variations and modifications may be effected by those skilled in the art without departing from the spirit and scope of the novel concepts of the disclosure.

The drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts of the same embodiment.

Drawings

FIGS. 1A to 1E are photomicrographs showing minced umbilical cord tissue mass-derived cells treated with only enzyme without cryopreservation, treated with enzyme prior to cryopreservation, not treated with enzyme nor cryopreserved, treated with enzyme after cryopreservation, and only cryopreserved without enzyme, respectively.

FIGS. 2A to 2C show the results of Fluorescence Activated Cell Sorting (FACS) or flow cytometry analysis by enzymatically treating the minced umbilical cord tissue mass-derived cells prior to cryopreservation, after cryopreservation and after enzymatic treatment, and after cryopreservation only without enzymatic treatment.

FIG. 3 shows the number of cells derived from a minced umbilical cord tissue piece, wherein the minced umbilical cord tissue piece is not subjected to the enzyme treatment and is subjected to the enzyme treatment before or after cryopreservation, respectively.

FIG. 4 shows the doubling times of minced umbilical cord tissue block-derived cells without and after cryopreservation.

FIG. 5 is a photomicrograph showing cell cultures of minced umbilical cord tissue pieces (left panel) and filtrate (right panel), respectively.

FIGS. 6A and 6B show the number of colonies and the number of cells derived from pieces of minced umbilical cord tissue and non-minced umbilical cord tissue, respectively.

FIG. 7 shows the results of RT-PCR analysis of stem cell marker genes.

FIG. 8A is a photomicrograph showing the cobblestone architecture in a co-culture of minced umbilical cord-derived stem cells and hematopoietic stem cells.

FIG. 8B shows the results of RT-PCR analysis, which is the signal pathway on umbilical cord tissue-derived stem cells associated with cell proliferation.

FIGS. 9A to 9C are micrographs showing adipocytes, chondrocytes, and vascular endothelial cells differentiated from minced umbilical cord tissue-derived stem cells, respectively.

FIGS. 10A to 10F show the results of the analysis of cells by Fluorescence Activated Cell Sorting (FACS) or flow cytometry before (FIGS. 10A to 10C) and after (FIGS. 10D to 10F) induction of angiogenesis.

FIG. 11 shows the number of stem cell colony forming units as a function of the length of time the cord tissue was incubated with the cryoprotectant solution.

Detailed Description

Definition of

The terms used in the specification of the present application have substantially the same meaning as commonly understood in the art to which they pertain within the scope of the present invention and the particular context of each term. Certain terms used in the following description to describe the present invention will be described below or elsewhere in the specification to provide those skilled in the art with an understanding of the relevant description of the present invention. For ease of reading, certain terms may be highlighted in italics and/or quotation marks, but use of these highlighted formats does not affect the scope or meaning of the terms. The same terms are used in the same context and have the same meaning regardless of whether they are in the highlighted format. In addition, the representation method of the same thing is more than one. Thus, terms discussed herein can be substituted for alternative terms and synonyms, and whether a term is detailed or discussed herein is not meant in any particular sense. Synonyms for certain terms are provided herein, but the use of one or more synonyms is not meant to exclude other synonyms. The examples provided in this specification, including the example terms discussed herein, are for illustrative purposes only and are not intended to limit the scope or meaning of the invention or any exemplified terms. Likewise, the invention is not limited to the various embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control.

As used herein, "about" or "approximately" refers to a value or range within 20%, preferably within 10%, and more preferably within 5% of a known value or range. The numerical values set forth herein are approximate, i.e., carry the meaning of the term unless the word "left or right," "about," or "approximately" is expressly written.

As used herein, the terms "cord tissue mass" and "minced cord tissue" are used interchangeably. A minced umbilical cord tissue mass refers to a piece of umbilical cord tissue that is less than 0.5 centimeters (cm) in size. In this context, the size of the umbilical cord tissue mass is no more than 0.4 cm, or no more than 0.3 cm, or no more than 0.2 cm. The size of the umbilical cord tissue mass not exceeding 0.2 cm means that the umbilical cord tissue mass can pass through a cell filter having a mesh size of 2 millimeters (mm).

As used herein, a "section of umbilical cord tissue" refers to a portion of the umbilical cord that is 0.5 centimeters or more than 0.5 centimeters in length. A length of umbilical cord tissue may be 0.5, 1, 2, or 3 centimeters, and even more than 3 centimeters.

The term "cryopreservation" or "cryopreservation" refers to the same method in which cells or whole tissues are cooled to a low temperature of below zero, usually 77K or below 196 ℃ (the boiling point of liquid nitrogen) for the purpose of preserving the cells or whole tissues. At these low temperatures, any biological activity, including biochemical reactions that lead to cell death, can be effectively suppressed. However, without the use of cryoprotectant solutions, the preserved cells tend to be damaged by freezing during the approach to low temperatures or warming back to room temperature.

The terms "freezing" and "cryopreservation" are used interchangeably.

The terms "frozen composition", "frozen solution", "cryopreservation composition" and "anti-freeze solution" are used interchangeably.

Unless otherwise specified, the terms "umbilical cord tissue-derived cells", "umbilical cord tissue mass-derived cells", "umbilical cord tissue-derived stem cells" and "umbilical cord tissue-derived MSCs" are interchangeable.

"doubling time" refers to the time required to double the size or value.

In one embodiment, the present invention is directed to a method of preserving an umbilical cord. The method comprises the following steps: obtaining a section of umbilical cord; mincing the umbilical cord into umbilical cord tissue pieces, wherein each umbilical cord tissue piece is no greater than 2 millimeters in size; mixing the umbilical cord tissue mass with a freezing composition to form a mixture, wherein the freezing composition comprises a cryoprotectant and a protein; shaking the mixture for a period of not less than 28 minutes but not more than 32 minutes; the mixture was then stored frozen.

In another embodiment, the invention is directed to a method of preserving an umbilical cord comprising: obtaining a section of umbilical cord; mincing the umbilical cord into umbilical cord tissue pieces; mixing the umbilical cord tissue mass with a freezing composition to form a mixture, wherein the freezing composition comprises a cryoprotectant and a protein; shaking the mixture for a period of not less than 20 minutes but not more than 40 minutes; the mixture was then stored frozen.

In another embodiment, the invention is directed to a method of obtaining umbilical cord tissue-derived stem cells from a section of umbilical cord comprising: obtaining a section of umbilical cord; mincing the umbilical cord into umbilical cord tissue pieces; mixing the umbilical cord tissue mass with a freezing composition to form a mixture, wherein the freezing composition comprises a cryoprotectant and a protein; shaking the mixture for a period of not less than 20 minutes but not more than 40 minutes; freezing and preserving the mixture; thawing the mixture and removing the frozen composition; the umbilical cord tissue pieces are then cultured in a culture medium to obtain umbilical cord-derived stem cells.

In one embodiment of the invention, the culture medium comprises human cord blood serum, or alternatively, the culture medium is comprised of DMEM and human cord blood serum.

In another embodiment of the invention, each umbilical cord tissue mass has a size of 2 millimeters or less (i.e., no more than 2 millimeters) than 2 millimeters.

In another embodiment of the present invention, the above method comprises continuously shaking the mixture for 25 to 30 minutes.

In another embodiment of the invention, the shaking step lasts 28 to 32 minutes. The shaking step may last for 30 minutes.

In another embodiment of the invention, the shaking step is performed at a temperature below 15 ℃, or between 0 ℃ and 10 ℃, or not above 4 ℃.

In another embodiment of the invention, the umbilical cord tissue mass is not treated by enzymatic digestion.

In another embodiment of the invention, the cord tissue mass is subjected to enzymatic digestion prior to the mixing step or after the thawing step. The method may further comprise inhibiting enzymatic digestion with an enzyme inhibitor.

In another embodiment, the present invention is directed to a frozen composition comprising: a) serum albumin or human serum; and b) a cryoprotectant, wherein the composition is free of non-human animal derived components.

In one embodiment of the present invention, the cryoprotectant is selected from at least one of dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol.

In another embodiment of the present invention, the cryopreservation solution comprises DMEM or phosphate buffered solution, and DMSO or glycerol.

In another embodiment of the present invention, the cryopreservation solution comprises DMEM or phosphate buffered solution, and DMSO or glycerol, and human umbilical cord blood serum or fetal bovine serum.

In another embodiment of the invention, the cryopreservation solution comprises DMEM or phosphate buffered solution, and DMSO or glycerol, and serum albumin or Plasma Protein Fraction (PPF).

Alternatively, the cryopreservation solution is composed of DMEM, DMSO, dextran, and human umbilical cord blood serum or serum albumin.

In another embodiment of the present invention, the protein component is selected from at least one of serum albumin, human serum and Plasma Protein Fraction (PPF).

In yet another embodiment of the invention, the frozen composition is free of non-human animal derived components and monosaccharides.

In yet another embodiment, the invention relates to a frozen composition comprising 30% to 50% human umbilical cord blood serum or 2.5% to 25% serum albumin; 5.5% to 55% DMSO; and 0.5% to 5% dextran.

In one embodiment of the invention, the frozen composition comprises 40% human umbilical cord blood serum or 2.5% to 25% serum albumin; 5.5% to 55% DMSO; and 0.5% to 5% dextran.

Examples

The following are merely exemplary of the devices, apparatus, methods and results associated with embodiments of the invention and should not be construed as limiting the invention. Please note that the titles or subtitles of each example are only used for convenience in reading and are not considered to be limitations of the present invention. Furthermore, while several theories are presented and disclosed herein, they are not to be considered limitations on the present invention, whether correct or not, as long as the invention is practiced by itself, and not in accordance with a particular theory or implementation.

Materials and methods

Cell culture medium

Commercially available media suitable for culturing umbilical cord-derived stem cells include, but are not limited to, RPMI, IMDM, DMEM, alpha-MEM, F12K, McCoy's 5a, and X-VIVO 10. One embodiment of the invention is the culturing of umbilical cord-derived cells in DMEM (90% to 70%) with human serum (10% to 30%).

In certain embodiments, cells, such as HUVEC, MS-5, or human stromal cell lines, are first cultured in commercially available media to obtain culture media containing the factors secreted by the cells. The medium containing the factors secreted by the cells is collected and used, either diluted or undiluted, to replace commercially available media for cell culture.

In other embodiments, commercially available media contain cell culture additives such as FBS, FCS, etc.; human peripheral blood serum; human umbilical cord blood serum; platelet Rich Plasma (PRP); cell growth factors such as IL-3, IL-6, TPO, FltL-3, SCF, EGF, TGF-. beta.bFGF; sodium pyruvate; glucose; glutamine amide and/or HEPES.

Alternatively, commercially available media for culturing umbilical cord-derived stem cells contain both cell culture additives and the above-described factors secreted by the cells.

Umbilical cord treatment

The cord was cleaned with alcohol (70% to 75%) and phosphate buffer. The cord is cut into appropriate pieces, which are then chopped into smaller pieces of cord tissue with mechanical tools. All tools were sterilized.

Enzyme treatment

To assess the effect of enzymatic digestion on the growth status of umbilical cord tissue-derived cells, the inventors performed the following experiments. A portion of the cord tissue mass was not enzymatically treated, and a portion of the cord tissue mass was enzymatically treated for 30 minutes before or after cryopreservation. The test was carried out by enzymatic digestion with trypsin or with a mixture of trypsin and collagenase. Other useful enzymes include, but are not limited to, proteases, gelatase, hyaluronidase, and any combination thereof. Digestion is stopped by enzyme inhibitors such as human serum (cord blood serum or non-cord blood serum) or fetal bovine serum. Other enzyme inhibitors that may be used in place of serum include, but are not limited to, trypsin inhibitors, serum-containing media (human or non-human animal serum), or buffered solutions containing EDTA. The cells were further washed several times with buffer solution or culture medium to remove the enzyme, and then cultured in the culture medium for 7 days with medium replacement every 3 to 7 days.

Freezing preservation

The umbilical cord tissue mass, with or without enzymatic digestion, is washed with phosphate buffer solution, mixed with the frozen composition, and then continuously shaken with a rotator (shaker) at a low temperature such as 4 ℃ for 30 minutes, and then washed with a solution of sodium chloride and sodium chlorideThe robot type liquid nitrogen freezing preservation and storage system is matched to store the liquid nitrogen at the temperature of about 196 ℃ below zero for at least one week so as to ensure the long-term viability of the liquid nitrogen freezing preservation and storage system. The freezing solution contains a buffer solution or a cell culture medium, and a cryoprotectant. The cryopreservation solution may optionally contain human-derived serum (e.g., human umbilical cord blood serum or human serum) and human-derived protein, which may be, but is not limited to, serum albumin or Plasma Protein Fraction (PPF). The buffer solution may be a phosphate buffer solution. The cell culture medium may be DMEM. The cryoprotectant may be dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, or propylene glycol. The cryopreservation solution may further comprise sucrose, trehalose or dextran.

To assess what effect frozen storage has on the culture and growth of cord-derived cells from umbilical cord tissue, the above umbilical cord tissue pieces were thawed and placed in a petri dish containing media and cultured in an incubator at 37 ℃ and 5% carbon dioxide.

Cell counting

The umbilical cord tissue pieces are cultured in culture medium for 7 days, replaced with new medium and cultured for another 8 to 14 days. After the cells were collected, the number of cells was counted. The cell population doubling time was calculated according to the following formula: doubling time is the number of culture hours/Log 2 (fold expansion of cells).

Co-culture of umbilical cord tissue-derived cells with Hematopoietic Stem Cells (HSCs)

The hematopoietic stem cells may be from human umbilical cord blood or peripheral blood. Using FICOLL-PAQUE^TMPLUS (GE healthcare group) isolated monocytes from human umbilical cord blood, followed by purification and isolation of CD34+ hematopoietic stem cells by magnetic field activated cell sorting (MACS). On the other hand, umbilical cord tissue piece-derived stem cells (i.e., MSCs) were seeded into 6-well plates, and then cultured in an incubator at 37 ℃ and 95% humidity for several days. Following replacement of the medium with new medium containing growth factors, umbilical cord tissue mass-derived stem cells were co-cultured with CD34+ Hematopoietic Stem Cells (HSCs) for one week. The medium was changed or more growth factors were added every 2 to 3 days. Growth factors include, but are not limited to, Stem Cell Factor (SCF), Thrombopoietin (TPO), and the Flt3-Ligand (Flt 3-Ligand). Finally, cell counting was performed with Trypan blue (Trypan blue, Invitrogen) and a hemocytometer.

Reverse transcription polymerase chain reaction (RT-PCR)

RT-PCR is used to examine the expression of early marker genes in stem cells and to detect the expression of genes in signal pathways associated with cell proliferation. When testing, useMini-kits (Qiagen corporation) and total RNA was extracted from umbilical cord tissue-derived cells in the manner specified in the manufacturer's manual.

Adipocyte differentiation

After the minced umbilical cord tissue-derived cells were grown to 100% confluence in DMEM/serum medium, they were cultured in serum-free culture medium for 48 hours and then cultured in adipocyte induction medium for another 4 weeks. The adipocyte induction medium contains DMEM, serum, antibiotics (streptomycin and penicillin), isobutylmethylxanthine, dexamethasone (dexamethasone), insulin and indomethacin. To determine the effect of adipocyte induction, the differentiated cells were stained with oil red o (oil red o). The positive adipocytes were stained red under an optical microscope.

Cartilage differentiation

The minced umbilical cord tissue-derived cells grown in the above-described manner in DMEM/serum medium were collected and then cultured in chondrocyte differentiation medium for 2 to 3 weeks, with replacement of new differentiation-inducing medium every 3 days. Chondrocyte differentiation media contained DMEM/serum, antibiotics (streptomycin and penicillin), dexamethasone, sodium pyruvate, transforming growth factor-beta 3 (TGF-beta 3). During chondrocyte induction for 14 days, the cells secrete an extracellular matrix containing type ii collagen, proteoglycans and anionic proteoglycans. The differentiated cartilage tissue was frozen, sectioned, and washed with an acetic acid solution, and then stained with Alcian blue (Alcian blue). The specimen sections were then counterstained with Nuclear fast red (Nuclear fast red). Differentiation of stem cells into chondrocytes is characterized by the appearance of zones of alcin staining. Strongly acidic sulfate mucilage is dyed blue, the nucleus is dyed pink to red, and the cytoplasm is dyed light pink.

Angiogenic differentiation

Cells were seeded on a plate coated with matrigel or collagen, and then cultured in endothelial growth medium-2 (EGM-2) to differentiate into vascular endothelial cells.

Length of time umbilical cord tissue pieces are cultured in freezing solution

To find the optimal length of time for the freezing solution to incubate, the cord tissue pieces are mixed with the freezing solution and gently shaken in a rotator in the cell incubator, but the shaking time varies. Briefly, a total of 3 donor umbilicals were collected at the time of the experiment, 14 cm each. After the umbilical cord tissue was evenly minced into small pieces (less than or equal to 2 mm), 7 equal samples were divided. Samples were taken from each sample every 10 minutes over a period of 60 minutes, added sequentially to a 15 ml centrifuge tube, mixed with the freezing solution, and shaken with a rotator. At the end of 60 minutes, all 7 samples were cryopreserved simultaneously by standard cryopreservation methods. 7 samples were thawed simultaneously after cryopreservation for 3 days, and the freezing solution was removed, followed by culturing the umbilical cord tissue pieces for 10 days. After 10 days of culture, the number of colony forming units and the number of umbilical cord tissue-derived stem cells from each sample were counted.

Results

The umbilical cord tissue mass-derived cells are Mesenchymal Stem Cells (MSCs). FIGS. 1A to 1D show umbilical cord tissue-derived cells on cell culture at day 7 of culture of the umbilical cord tissue pieces, respectively. The cells are spindle-shaped and proliferate rapidly. Prior to cell culture to obtain umbilical cord tissue-derived cells, each piece of umbilical cord tissue is either subjected to only enzymatic digestion (FIG. 1A), or is subjected to both enzymatic digestion and cryopreservation (FIG. 1B), or is a new piece of umbilical cord tissue that has not been subjected to either enzymatic or cryopreservation (FIG. 1C), or is subjected to cryopreservation first and then enzymatic digestion (FIG. 1D), or is subjected to only cryopreservation (FIG. 1E). The results show that cryopreservation and/or enzymatic treatment did not affect the shape of the umbilical cord tissue mass-derived cells.

The cell types of the umbilical cord tissue-derived cells are evaluated by FACS (fluorescence activated cell sorting) or flow cytometry. For the analysis of cell surface antigens, umbilical cord tissue mass-derived cells were collected, washed with phosphate buffer solution, and then reacted with antibodies CD13, CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, HLA-ABC, HLA-DR, 7AAD or SSEA-4, respectively, followed by coupling with Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE) or phycoerythrin cyanine dye 5(PE-Cy5) (BD biosciences). Fig. 2A to 2C are cell scattergrams. Prior to culturing the umbilical cord tissue block graft, the cells are derived from umbilical cord tissue block grafts that were subjected to enzymatic digestion prior to (FIG. 2A) or after cryopreservation (FIG. 2B), or were subjected to cryopreservation only without enzymatic treatment (FIG. 2C), respectively. Each dot represents a single cell, and its location represents its Forward Scatter (FSC) intensity value (cell size) and Side Scatter (SSC) intensity value (cell granularity). Forward scatter is roughly proportional to cell diameter, while side scatter is proportional to cell size. The results show that pre-treatment of the cord tissue mass with enzymes and cryopreservation does not affect the purity and properties of cord tissue mass-derived cells according to the methods of the invention. For the analysis of cell surface antigens, umbilical cord tissue mass-derived cells were collected, washed with phosphate buffer solution, and then reacted with antibodies CD13, CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, HLA-ABC, HLA-DR, 7AAD or SSEA-4, respectively, followed by coupling with Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE) or phycoerythrin cyanine dye 5(PE-Cy5) (BD biosciences). The results show that the type of antigen that can be identified on the cell surface is the same whether the cell line is derived from cord tissue pieces that have been enzymatically treated before or after cryopreservation, or cord tissue pieces that have not been enzymatically treated before or after cryopreservation (relevant data cannot be shown).

The inventors also assessed the effect of enzyme treatment on the number of cord tissue-derived cells before and after cryopreservation of the cord tissue pieces, and the experimental data are expressed in relative cell numbers (%), where 100% is defined as the number of cells derived from a cord tissue piece that had been subjected to enzyme digestion prior to cryopreservation and then thawed (as in the "pre-freezing enzyme treatment" column of FIG. 3). FIG. 3 shows the cell number of whether the umbilical cord tissue mass received enzyme treatment (e.g., the "non-enzyme treated" column of FIG. 3), or whether the umbilical cord tissue mass was treated with enzyme before or after cryopreservation (e.g., the "pre-freezing enzyme treated" column and the "post-freezing enzyme treated" column of FIG. 3). Furthermore, substitution of trypsin inhibitors for serum as enzyme inhibitors to stop enzymatic digestion did not significantly affect the number of cells collected (no relevant data shown). In a control study, only one cord tissue was used for each set of experiments.

The inventors also evaluated the effect of cryopreservation on cell doubling time by comparing the doubling time of cells derived from a cord tissue mass prior to cryopreservation (i.e., without cryopreservation treatment) to the doubling time of cells derived from a cord tissue mass after one week of cryopreservation. The results show that cryopreservation of the umbilical cord tissue pieces according to the invention has no significant effect on cell doubling time (figure 4). Thus, the freezing and storage process of the present invention does not adversely affect the yield of cord tissue mass-derived MSCs.

The inventors also analyzed whether replacement of serum in the freezing solution with serum albumin would have an effect on the yield of umbilical cord tissue mass-derived cells. The results show that there was no significant difference in cell numbers when serum albumin was substituted for serum in the frozen composition. The significance of this finding is to provide a serum-free and viable frozen composition.

The results of the experiments in FIG. 5 show that the cells grown and proliferated in the tissue culture plates are derived from the umbilical cord tissue mass, not from free cells in the umbilical cord tissue sample. The umbilical cord tissue mass is mixed with the buffer solution and then filtered through a Cell filter (BDFALCON) with a mesh size of 100 microns^TM) And (5) filtering. The pieces of umbilical cord tissue passed through the mesh are separately cultured with a buffer solution (i.e., filtrate or passed through a fluid) in a culture medium suitable for the umbilical cord tissue-derived cells. The results show that only cultures containing umbilical cord tissue pieces show cell growth and migration, while culture dishes containing only the filtrate do not. In other words, according to the present invention, the cells derived from the umbilical cord tissue mass (i.e., mesenchymal stem cells) are not from free cells that are free to swim around, but from stem cells within the umbilical cord tissue. In other words, stem cells are produced from the umbilical cord tissue mass and migrate out of the tissue implant under culture conditions.

To assess whether the act of mincing had an effect on the growth of umbilical cord tissue-derived stem cells, the inventors directly mixed a section of umbilical cord of appropriate length (about 2 cm) without mincing with a freezing solution, followed by cryopreservation at-160 ℃ for 5 to 7 days and culture after thawing, during which umbilical cord tissue-derived cells could grow. For comparison, another section of homologous umbilical cord is cut up, mixed with the freezing solution, frozen and preserved under the same conditions, and then thawed and cultured. Both experiments were observed microscopically for cell growth. The results show that both experiments produced cells regardless of whether the umbilical cord tissue was minced or not.

The number of cell populations on the cell culture was counted at day 10 of culture. As shown in FIG. 6A, the number of cell populations derived from the minced umbilical cord tissue pieces was 20.5 times the number of cell populations derived from the uncut portions of umbilical cord tissue. The number of cell populations is calculated based on the number of umbilical cord tissue blocks in which the stem cell migration phenomenon occurs. In other words, the number of umbilical cord tissue blocks in which the stem cell migration phenomenon occurs is the number of cell populations. As shown in FIG. 6B, the relative cell number of cells derived from the minced umbilical cord tissue pieces was 11.17 times the relative cell number of cells derived from the uncut umbilical cord tissue portions. The results show that chopping cord tissue into small pieces affects the number of stem cells produced by the cord tissue. Thus, the results show that cord tissue can produce far more cord tissue-derived stem cells after being minced and cryopreserved in the manner of the present invention than cord tissue-derived stem cells produced after the non-minced cord tissue is cryopreserved. This finding is of great clinical significance, as sufficient stem cells must be provided in a short time to minimize the risk of cell therapy.

The umbilical cord tissue-derived cells all express pluripotent stem cell markers such as BMP4, Oct4, REX1, SOX2, Nanog and the like. The agarose gel bands shown in FIG. 7 are DNA products of RT-PCR showing gene expression of the stem cell markers in cells derived from minced umbilical cord tissue. The results show that stem cells derived from minced umbilical cord tissue in the manner of the present invention are pluripotent, naive stem cells.

According to the present invention, stem cells derived from minced umbilical cord tissue can be used as feeder cells to support the growth and proliferation of Hematopoietic Stem Cells (HSCs). The co-culture of umbilical cord tissue-derived cells and HSCs formed a cobblestone structure (FIG. 8A) in which each cobblestone was a mass of HSC cells that appeared as dark cobblestones in a phase contrast microscope. The principle of forming a cobble structure is as follows: HSCs that float loosely on feeder cells are spherical and thus refractive, while HSCs located below feeder cells are flat and thus not refractive. The results show that the hematopoietic stem cells can be cultured together with umbilical cord tissue mass-derived stem cells to promote the growth of hematopoietic stem cells.

The inventors also detected signaling pathways associated with cell proliferation in umbilical cord tissue-derived stem cells obtained according to the present invention. The detected signal is shown in fig. 8B, and includes: IGF-2 signaling pathways, such as IGF-2 and IGFBP 5; FGF signaling pathways, such as FGF-1 and FGF-2; gap junction signaling pathways, such as Panx-1, Panx-2; EMT pathways such as α -SMA, osteopontin, CK18, Twist, BMP-4, TGF- β 1; early genes, such as Oct4, Rex1, SOX 2; other genes, such as angiopoietin-like-2 (Angptl-2), dlk; wnt signaling pathways, such as Wnt5a, SFRP1, Dkk 3; surface markers, such as CD 13; and adhesion molecules such as N-cadherin (N-cadherin), beta-catenin (beta-catenin).

Cells derived from minced umbilical cord tissue are pluripotent stem cells that have the ability to form fat, cartilage, and angiogenesis and neovascularization. FIG. 9A shows adipocytes differentiated from umbilical cord tissue-derived stem cells and stained with oil red after 4 weeks of culture in an adipose medium. Culturing in cartilage medium for 2 to 3 weeks induced differentiation of umbilical cord tissue-derived stem cells into chondrocytes, which were positive after staining with alcian blue (fig. 9B). Endothelial growth medium-2 (EGM-2) then induces differentiation of umbilical cord tissue-derived stem cells into vascular endothelial cells (fig. 9C), where the differentiated cells become elongated and link to each other, eventually forming a capillary network.

Flow cytometry is used to detect changes in cell size (diameter), particle size and surface antigens before and after induction of angiogenesis. FIGS. 10A and 10D show the changes in cell size and particle size. In addition, the induced differentiated cells were positive after staining with anti-VEGF-R1 and anti-VE-Cad antibodies (FIGS. 10B, 10C, 10E and 10F), indicating that the differentiated cells have characteristics of vascular endothelial cells.

The ability of cryopreserved umbilical cord tissue pieces to produce umbilical cord-derived stem cells is influenced by the length of time the umbilical cord tissue is cultured in the freezing solution. FIG. 11 shows the number of Colony Forming Units (CFU) as a function of the incubation time of the freezing solution. After each sample was incubated with the frozen solution for different periods of time, the CFU of each sample was compared with the sample incubated for 30 minutes. A total of 3 donor samples (n-3) were used in the experiment. The P value ≦ 0.05 is indicated by an asterisk. Bars show mean ± SEM. The results show that the incubation with the frozen solution is suitably continued for 20 to 40 minutes, and the optimum incubation time is 30 minutes. The umbilical cord tissue pieces cultured with only the frozen solution for 0 to 20 minutes were not sufficiently attached to the bottom surface of the culture dish. If the umbilical cord tissue block can be attached to the bottom surface of the culture dish, the MSC can be more easily attached to the bottom surface of the culture dish so as to be beneficial to growth. The yields of umbilical cord tissue block-derived MSCs were unstable in 40 to 60 min of umbilical cord tissue blocks cultured in frozen solution due to the rather large difference in cell numbers in the repeated experiments (relevant data cannot be shown).

The above embodiments are merely exemplary, and are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations of the present invention are possible in light of the above teachings.

The embodiments and examples were chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Numerous alternative embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the disclosure. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description and the examples described therein.

In the above description of the present invention, several references, including patents, patent applications, and various publications, have been cited. Citation and/or discussion of such references is intended as an illustration of the present invention and is not an admission that such references are "background" to the present invention. All references cited and discussed in this patent specification are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually incorporated by reference.

Claims (8)

1. A method of preserving umbilical cord tissue comprising the steps of:

obtaining a section of umbilical cord;

mincing the umbilical cord into umbilical cord tissue pieces, wherein each umbilical cord tissue piece has a size of 2 millimeters or less than 2 millimeters;

mixing the umbilical cord tissue pieces with a freezing composition to form a mixture, wherein the freezing composition comprises:

(a) 30% to 50% human umbilical cord blood serum or 2.5% to 25% serum albumin;

(b) 5.5% to 55% dimethyl sulfoxide; and

(c) 0.5% to 5% dextran;

shaking the mixture, said shaking step being performed at a temperature below 15 ℃ and for a period of not less than 20 minutes but not more than 40 minutes; and

freezing and storing the mixture,

Wherein the umbilical cord tissue pieces are subjected to an enzymatic digestion treatment prior to the mixing step.

2. The method of claim 1, comprising shaking the mixture for 25 to 35 minutes.

3. The method of claim 1, wherein the shaking step is performed at a temperature between 0 ℃ and 10 ℃.

4. The method of claim 1, wherein the shaking step is carried out at a temperature not exceeding 4 ℃.

5. The method of claim 1, wherein the shaking step is performed for 28 to 32 minutes.

6. A method for obtaining umbilical cord tissue-derived stem cells from a section of umbilical cord comprising the steps of:

obtaining a section of umbilical cord;

mincing the umbilical cord into umbilical cord tissue pieces, wherein each umbilical cord tissue piece has a size of 2 millimeters or less than 2 millimeters;

mixing the umbilical cord tissue pieces with a freezing composition to form a mixture, wherein the freezing composition comprises:

(a) 30% to 50% human umbilical cord blood serum or 2.5% to 25% serum albumin;

(b) 5.5% to 55% dimethyl sulfoxide; and

(c) 0.5% to 5% dextran;

shaking the mixture, said shaking step being performed at a temperature below 15 ℃ and for a period of not less than 20 minutes but not more than 40 minutes;

freezing and preserving the mixture;

thawing the mixture and removing the frozen composition; and

culturing the umbilical cord tissue mass in a culture medium to obtain umbilical cord tissue-derived stem cells,

Wherein the umbilical cord tissue pieces are subjected to an enzymatic digestion treatment prior to the mixing step.

7. The method of claim 6, comprising shaking the mixture for 25 to 35 minutes.

8. A method for obtaining umbilical cord tissue-derived stem cells from a section of umbilical cord comprising the steps of:

obtaining a section of umbilical cord;

mincing the umbilical cord into umbilical cord tissue pieces, wherein each umbilical cord tissue piece has a size of 2 millimeters or less than 2 millimeters;

mixing the umbilical cord tissue pieces with a freezing composition to form a mixture, wherein the freezing composition comprises:

(a) 30% to 50% human umbilical cord blood serum or 2.5% to 25% serum albumin;

(b) 5.5% to 55% dimethyl sulfoxide; and

(c) 0.5% to 5% dextran;

shaking the mixture, said shaking step being performed at a temperature below 15 ℃ and for a period of not less than 20 minutes but not more than 40 minutes;

freezing and preserving the mixture;

thawing the mixture and removing the frozen composition; and

culturing the umbilical cord tissue mass in a culture medium to obtain umbilical cord tissue-derived stem cells, wherein the umbilical cord tissue mass has not been treated by enzymatic digestion.