CN112251400A - Enzyme-free pretreatment and method for culturing adipose-derived mesenchymal stem cells - Google Patents
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Abstract
The invention relates to the field of cell culture, in particular to a method for enzyme-free pretreatment and culture of adipose-derived mesenchymal stem cells. The method for enzyme-free pretreatment and culture of adipose-derived mesenchymal stem cells comprises the following steps: a. collecting adipose tissue; b. dissolving mature fat in the adipose tissue by a fat-soluble composition; the fat-soluble composition is free of enzymes; c. centrifuging to separate solid and liquid fractions of the adipose tissue of the solubilized mature fat; d. dividing the solid portion of the adipose tissue into explant pieces; e. drying the explant pieces; f. immersing the explant in an adipose-derived mesenchymal stem cell culture medium to form an explant culture composition and culturing; g. obtaining a target adipose-derived mesenchymal stem cell from the explant culture composition. The method can obtain a considerable amount of adipose-derived mesenchymal stem cells in an in vitro culture environment, and is easy, safe, rapid, cheap and more effective.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a method for enzyme-free pretreatment and culture of adipose-derived mesenchymal stem cells.
Background
Mesenchymal stem cells were initially found in bone marrow and subsequently have also been demonstrated to contain mesenchymal stem cells in a variety of tissues, including subcutaneous adipose tissue, where adipose tissue is a rich source of mesenchymal stem cells and is relatively readily available and suitable for tissue engineering and regenerative medicine applications. Many documents demonstrate the pluripotency of Adipose-Derived mesenchymal stem Cells (ADSCs), which have the potential to differentiate into cartilage, hard bone and adipocytes.
The rodcell et al, in 1960, created a traditional, and at the same time most widely used, method of isolating Stromal Vascular Fraction (SVF) from adipose tissue and culturing expanded mesenchymal stem cells in vitro, originally for isolating Adipocytes (Adipocytes) in adipose tissue. The method comprises the steps of cutting a Fat pad (Fat pad) of a rat, washing most blood cells, adding collagen hydrolase (collagen) to decompose tissues, separating a mature Fat cell group from a precipitate of a stromal vascular part in a centrifugation mode, and culturing and amplifying the stromal vascular part to obtain the adipose interstitial stem cells. The stromal vascular fraction is a heterogeneous cell population (heterogeneous cell population) comprising different cell types such as Blood cells (Blood cells), Blood stem cells (hematonic stem cells), Fibroblasts (Fibroblasts), peripheral cells (Pericytes), Endothelial cells (Endothelial cells), adipose precursor cells (Pre-adipocytes), and mesenchymal stem cells (ADSCs), and then treated with a red Blood cell lysis buffer (rbclys buffer) to lyse and remove red Blood cells from the stromal vascular fraction; therefore, adipose stromal stem cells can be isolated and expanded from mesenchymal stem cells based on their ability to adhere to the surface of a cell culture dish.
Emerging regenerative medicine fields require a reliable source of stem cells. Adipose tissue has proven to be a rich source of adult pluripotent stem cells and is relatively easy to obtain, consistent with the properties of tissue engineering and regenerative medicine applications. The current separation mode of the stromal vascular fraction requires the use of collagenase, in a concentration range of 0.075-0.1%; among them, the collagen-hydrolyzing enzymes are derived from Clostridium histolyticum (Clostridium histolyticum) whose decomposition efficiency varies depending on the quality and concentration of the enzymes, such as Crude extract (raw extract) or purification (Purified), and the presence or absence of other proteolytic enzymes such as neutral proteolytic enzyme (Dispase) or thermophilic proteolytic enzyme (Thermolysin). However, the source and the residue of collagenase will cause safety concerns in the future of clinical application, so the Food and Drug Administration (FDA) does not approve the clinical application of producing adipose-derived mesenchymal stem cells by using collagenase.
In view of the above, there is a need to develop a practical method for culturing adipose-derived mesenchymal stem cells without using enzymes, and simultaneously increasing the expansion rate of adipose-derived mesenchymal stem cells.
Disclosure of Invention
Accordingly, an object of the present invention is to provide a method for enzyme-free pretreatment and culture of Adipose-derived stromal stem cells (ADSCs), comprising the following steps: a. collecting adipose tissue; b. dissolving mature fat in the adipose tissue with a fat-soluble composition (Lipppoissolve composition), wherein the fat-soluble composition does not contain enzymes; c. centrifuging to separate solid and liquid fractions of the adipose tissue of the solubilized mature fat; d. dividing the solid portion of the adipose tissue into explant pieces; e. subjecting the explant pieces to a drying step; f. immersing the Explant piece in an adipose-derived mesenchymal stem cell culture medium to form an Explant culture composition (Explant culture composition) and culturing; obtaining the adipose-derived mesenchymal stem cells of interest from the explant culture composition, and the fat-soluble composition of step b is free of enzymes.
In one embodiment of the present invention, the adipose stromal stem cells obtained in step g retain their original cellular characteristics.
In yet another embodiment of the present invention, the adipose tissue of step a is obtained from a Subcutaneous (Subcutaneous), Visceral fat pad (Visceral fat pad), or liposuction (Lipoaspirate) of a subject.
In another embodiment of the present invention, the explant culture composition of step f comprises a mesenchymal stem cell composition (Stromal cell composition), and the mesenchymal stem cell composition comprises a population of mesenchymal stem cells derived from adipose tissue of a mammal; and the mesenchymal stem cell population comprises a Blood cell (Blood cells), a Blood stem cell (Hematopoietic stem cells), a fibroblast cell (Fibroblasts), a peripheral cell (Pericytes), an Endothelial cell (Endothelial cells), a Pre-adipocyte (Pre-adipocyte), an adipocyte (adipocyte), and/or an mesenchymal stem cell (ADSCs).
In another embodiment of the present invention, the fat-soluble composition of step b comprises a physiological saline solution and a fat-soluble material capable of dissolving fat tissue; and the reaction time for dissolving the mature fat in the adipose tissue by the fat-soluble composition is 5-10 minutes; wherein the concentration of the physiological saline in the fat-soluble composition is 99 percent, and the concentration of the fat-soluble material capable of dissolving fat tissues is 1 percent; the fat-soluble material capable of dissolving the adipose tissues is essential Phospholipid (Phospholipid) and/or Phosphatidylcholine (Phosphatidylcholine); and the concentration of the essential phospholipid or the phosphatidylcholine in the fat-soluble material is 40-60mg/mL, wherein the concentration is preferably 50 mg/mL.
In yet another embodiment of the present invention, the explant of step d is prepared by cutting the solid portion of the adipose tissue into a tissue mass having a diameter of about 5 to 10 mm; and placed in a cell culture dish at a proper interval; the drying step of step e is drying the explant pieces for 20-30 minutes; and the adipose stromal stem cell culture medium of the step f comprises 89-97% of alpha MEM, 2-10% of Human Platelet Lysate (HPL), and 1% of antibiotics.
The method for enzyme-free pretreatment and culture of adipose-derived mesenchymal stem cells provided by the invention comprises the steps of extracting the mesenchymal stem cells from adipose tissues, wherein the mesenchymal stem cells have the characteristic of being attached to a culture dish for growth, but excessive fat molecules in the adipose tissues can block the attachment of the mesenchymal stem cells.
The method for enzyme-free pretreatment and culture of the adipose-derived stromal stem cells provided by the invention can obtain a considerable amount of adipose-derived stromal stem cells in an isolated culture environment, is easier, safer, faster, cheaper and more effective than the traditional method, and better conforms to the Good Tissue Practice (Good Tissue Practice) of the cell Tissue, so that the method for enzyme-free pretreatment and culture of the adipose-derived stromal stem cells can be more effectively applied to future Tissue engineering and regenerative medicine.
The following examples are presented to illustrate the present invention and are not to be construed as limiting the scope of the invention, which is intended to be limited only by the appended claims.
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FIG. 1 is a photograph of explant culture of adipose tissue of the present invention;
FIG. 2 is a photograph of adipose stromal stem cell growth of an explant that is adipose tissue of the present invention;
FIG. 3 is a bar graph of the total yield of stromal cells obtained for the method of the invention.
Detailed Description
As used herein, the numerical values are approximations and all numerical data are reported to be within the 20 percent range, preferably within the 10 percent range, and most preferably within the 5 percent range.
Statistical analysis was performed using Excel software. Data are presented as mean ± Standard Deviation (SD).
The invention provides a method for pretreating and culturing adipose-derived mesenchymal stem cells without enzyme, wherein the mesenchymal stem cells are extracted from adipose tissues, and have the characteristic of being attached to a culture dish for growth, but excessive fat molecules in the adipose tissues can prevent the attachment of the mesenchymal stem cells, so that the method uses an interface active agent naturally existing in a human body, such as essential phospholipid or phosphatidylcholine, to saponify and dissolve mature fat around the adipose tissues, so that the mesenchymal stem cells in the adipose tissues can be directly exposed on the culture dish, and the contact area between the mesenchymal stem cells and the culture dish is increased, thereby achieving better attachment rate and culture rate; and because the interface active agent naturally existing in the human body is used, the damage to mesenchymal stem cells in the process of dissolving fat can be reduced by being closer to the physiological environment.
Meanwhile, in the conventional method for separating mesenchymal stem cells from adipose tissues, collagen hydrolase (collagen) is required to separate mature adipocytes, but the damage of enzymes to the cells is not clear at present, for example, the source and the residue of the enzymes cause safety concerns in clinical application in the future; therefore, the invention provides a practical method for separating and culturing adipose-derived mesenchymal stem cells without using collagen hydrolase, which can directly and effectively reduce the safety doubt caused by using enzyme on mesenchymal stem cells and simultaneously achieve the purpose of improving the cell culture yield.
As used herein, the term "fat-soluble composition" is meant to encompass: a composition comprising 99% of physiological saline and 1% of fat-soluble material capable of dissolving adipose tissues; wherein, the fat-soluble material capable of dissolving the adipose tissue can be, but not limited to, essential Phospholipid (Phospholipid) and/or Phosphatidylcholine (Phosphatidylcholine), and the concentration of the essential Phospholipid or the Phosphatidylcholine in the fat-soluble material is 40-60mg/mL, wherein the concentration is preferably 50 mg/mL.
As used herein, the term "surfactant naturally present in the human body" can be, but is not limited to, an essential phospholipid or phosphatidylcholine; wherein, the phosphorylcholine is an endogenous storage form of Choline (Choline) in human bodies.
According to the invention, mature fat in adipose tissue is removed by using fat-soluble composition (Lippodissolve composition) containing phosphatidylcholine, so that the contact area and the attachment rate of the explant of the adipose tissue and the surface of a culture dish can be increased, and the exposure, migration and cell culture yield of mesenchymal stem cells in the adipose tissue are increased.
As used herein, the term "Explant culture" means an in vitro culture method that avoids the damage that collagen hydrolase may cause to mesenchymal stem cells and the possibility of remaining therein, to maximize the survival rate of mesenchymal stem cells; the explant culture method is beneficial to reproducing and retaining original characteristics (such as Extracellular matrix and Extracellular matrix) of mesenchymal stem cells in original tissues, such as Extracellular matrix (ECM) and Cell-Cell interaction (Cell-Cell interaction) of the mesenchymal stem cells, so that the cultured mesenchymal stem cells are healthier and are easier to be practically applied to human bodies.
The method for enzyme-free pretreatment and culture of the adipose-derived stromal stem cells provided by the invention can obtain a considerable amount of adipose-derived stromal stem cells in an isolated culture environment, is easier, safer, faster, cheaper and more effective than the traditional method, and better conforms to the Good Tissue Practice (Good Tissue Practice) of the cell Tissue, so that the method for enzyme-free pretreatment and culture of the adipose-derived stromal stem cells can be more effectively applied to future Tissue engineering and regenerative medicine.
The following will describe in detail the detailed method for enzyme-free pretreatment and amplification of adipose-derived mesenchymal stem cells according to the present invention, and the comparative test of the effects of the conventional method for separating and amplifying adipose-derived mesenchymal stem cells from adipose tissues in the present invention; compared with the traditional method, the enzyme-free pretreatment and culture method of the adipose-derived mesenchymal stem cells can directly and effectively reduce the safety doubt caused by using the enzyme to the mesenchymal stem cells, maximize the yield of the in-vitro culture of the mesenchymal stem cells, and better meet the excellent operation specification of cell tissues, so that the method can be more effectively applied to the fields of future tissue engineering and regenerative medicine.
Example 1 enzyme-free pretreatment and method for amplifying adipose-derived mesenchymal stem cells according to the present invention
One embodiment of the present invention is a method for enzyme-free pretreatment and amplification of adipose-derived mesenchymal stem cells; the following procedures were carried out in a BioSafety Cabinet (Biosafety cassette) except that the centrifugation step was carried out in a centrifuge and the cell culture step was carried out in a cell incubator. First, 10mL of adipose tissue was obtained from Subcutaneous (Subcutaneous), Visceral fat pad (viscerate pad), or liposuction (Lipoaspirate) of an individual, wherein the individual contained a mammal; then, the adipose tissue is treated with 1% of fat-soluble composition at room temperature for 5-10 min; wherein the fat-soluble composition comprises 99% of physiological saline and 1% of Phosphatidylcholine (Phosphatiddylcholine), the Phosphatidylcholine is a surfactant naturally existing in a human body, can gently dissolve mature fat in adipose tissues, and can gently centrifuge partial dissolved adipose tissues to separate solid and liquid parts of the adipose tissues, wherein the mild centrifugation condition is room temperature, the centrifugal force is 300 Xg, and the centrifugation lasts for 10 minutes; then, the disintegrated fat centrifugal sediment is cut into explant pieces with the diameter of about 5-10 mm by using sterile dissecting scissors, the explant pieces are transferred to a tissue culture dish by using sterile forceps and are dried, then an appropriate amount of fat mesenchymal stem cell culture medium is lightly covered on the explant, the explant pieces are partially soaked in the fat mesenchymal stem cell culture medium, and the culture medium is mixed with 5% of CO at 37 DEG C2The culture chamber of (1), wherein the adipose-derived mesenchymal stem cell culture medium contains 89-97% Alpha-Minimum Essential Media (α MEM, available from Hyclone under the number SH30265), 2-10% Human Platelet Lysate (HPL), and 1% antibiotic, preferably Penicillin/streptomycin; among them, the concentration of α MEM is preferably 94%, and the concentration of human platelet lysate is preferably 5%; followed byObserving the growth state of the adipose-derived mesenchymal stem cells in the explant by a microscope after 10 days of culture; and the total quantity of the mesenchymal cells obtained by the method is measured by a method of calculating the total quantity of the living cells by using a dead cell stain (Trypan blue) and a cell counting plate (Hemocytometer).
In addition, this embodiment is also compared with the conventional method for separating and amplifying adipose stromal stem cells from adipose tissue, i.e., the conventional method using collagenase; wherein the method comprises centrifuging 10mL of adipose tissue at 1500rpm for 10 min at room temperature, filtering off blood components, adding freshly prepared Collagenase (collagen) with 0.1% volume, treating in 37 deg.C shaking water bath for 60 min to decompose adipose tissue, centrifuging at 3000rpm at room temperature for 10 min, removing supernatant to obtain concentrated Stromal Vascular Fraction (SVF), dispersing and washing SVF with physiological test water, centrifuging again to obtain SVF, adding erythrocyte lysate (RBC lysate), treating in shaking water bath at room temperature for 10 min to remove erythrocytes, centrifuging again, filtering the undecomposed adipose tissue in SVF with Cell filter (Cell separator) with pore size of 70 μm, and filtering at 37 deg.C with 5% CO2The total number of the mesenchymal cells obtained by the traditional method is measured by a method of culturing the cells in the incubator, and also using a dead cell stain together with a cell counting plate to calculate the total number of the living cells.
The method for culturing the explant of adipose tissue according to the present invention is shown in FIG. 1, in which it can be seen that the explant pieces cut with sterile scissors to about 5-10 mm in diameter are uniformly distributed in the culture dish containing the adipose-derived mesenchymal stem cell culture medium; the growth of the adipose tissue-derived stem cells in the explants of adipose tissue of the present invention is shown in FIG. 2, in which a number of adipose tissue-derived stem cells growing from the left side of the explant to the right side can be seen; the results of the present invention showing that the adipose tissues in the non-enzyme treatment process have a higher yield of adipose stromal cells are shown in FIG. 3, wherein it can be seen that 5.42X10 is obtained after 10 days of the culture of the non-enzyme treatment process from 10mL of breast subcutaneous adipose tissues7Inter-roomCompared with the traditional enzyme treatment method, the total yield of the mesenchymal cells can be improved by about 28 times; these results show that the enzyme-free pretreatment and culture method of adipose-derived mesenchymal stem cells provided by the present invention can amplify a considerable amount of adipose-derived mesenchymal stem cells in an in vitro culture environment.
Wherein, the traditional enzyme culture method firstly uses collagenase for treatment to separate SVF, and then uses SVF to culture adipose-derived stem cells, and compared with the explant in the culture method, the components of the adipose-derived stem cells are reduced by most of adipose cells and blood cells; in the method for amplifying the adipose-derived mesenchymal stem cells by enzyme-free pretreatment, the mesenchymal stem cells are amplified by an external plant sheet, wherein cell interaction and extracellular matrix in adipose tissue agglomerates are reserved, and the method can reproduce and reserve the original characteristics of the mesenchymal stem cells in the original tissues, so that the cultured mesenchymal stem cells are healthier and are easier to be practically applied to a human body; and, wherein the use of a natural human body-resident interface active agent mildly removes mature fat in adipose tissue to increase the contact area and the adhesion rate of cells in adipose tissue to the surface of a culture dish, thereby increasing the exposure and migration of mesenchymal stem cells and the yield of cell culture inside adipose tissue while maintaining the original characteristics of mesenchymal stem cells, so that the yield of mesenchymal stem cells is higher than that of the original method.
In summary, the enzyme-free pretreatment and adipose-derived stromal stem cell culturing method provided by the invention can obtain a considerable amount of adipose-derived stromal stem cells in an in vitro culture environment, and compared with the conventional method, the method is easier, safer, faster, cheaper and more effective, and better meets the Good Tissue Practice (Good Tissue Practice) of the cell Tissue, so that the enzyme-free pretreatment and adipose-derived stromal stem cell culturing method can be more effectively applied to future Tissue engineering and regenerative medicine.
Claims (10)
1. A method for enzyme-free pretreatment and culture of adipose-derived mesenchymal stem cells comprises the following steps:
a. collecting adipose tissue;
b. dissolving mature fat in the adipose tissue by a fat-soluble composition;
c. centrifuging to separate solid and liquid fractions of adipose tissue from which mature fat has been solubilized;
d. dividing the solid portion of the adipose tissue into explant pieces;
e. drying the explant pieces;
f. immersing the explant in an adipose-derived mesenchymal stem cell culture medium to form an explant culture composition and culturing; and
g. obtaining a target adipose-derived mesenchymal stem cell from the explant culture composition;
wherein the fat-soluble composition of step b does not contain enzymes.
2. The method of claim 1, wherein the adipose stromal stem cells obtained in step g retain their original cellular characteristics.
3. The method of claim 1, wherein the adipose tissue of step a is taken from the subcutaneous, visceral fat pad, or lipoaspirate of a subject.
4. The method of claim 1, wherein the explant culture compositions of step f each comprise a mesenchymal stem cell composition comprising a population of mesenchymal stem cells derived from adipose tissue of a mammal.
5. The method of claim 3, wherein the population of mesenchymal stem cells comprises a blood cell, a blood stem cell, a fibroblast, a peripheral cell, an endothelial cell, an adipose precursor cell, an adipose cell, and/or a mesenchymal stem cell.
6. The method of claim 1, wherein the fat-soluble composition of step b comprises a physiological saline solution and a fat-soluble material capable of solubilizing adipose tissue.
7. The method according to claim 5, wherein the concentration of physiological saline in the fat-soluble composition is 99% and the concentration of the fat-soluble material capable of dissolving adipose tissue is 1%.
8. The method according to claim 5, wherein the fat-soluble material capable of dissolving adipose tissue is an essential phospholipid, and/or a phosphatidylcholine; and the concentration of the essential phospholipid or the phosphatidylcholine in the fat-soluble material is 40-60 mg/mL.
9. The method of claim 1, wherein the explant of step d is prepared by cutting the solid portion of said adipose tissue into a tissue mass having a diameter of about 5 to 10 mm.
10. The method of claim 1, wherein the adipose stromal stem cell culture medium of step f comprises 89-97% α MEM, 2-10% human platelet lysate, and 1% antibiotics.
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| US20090233885A1 (en) * | 2006-07-14 | 2009-09-17 | Duncan Diane I | Compositions and Methods for Fat Reduction |
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