CN103361299B - The method of separate stem cells and test kit - Google Patents
The method of separate stem cells and test kit Download PDFInfo
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- CN103361299B CN103361299B CN201210096966.0A CN201210096966A CN103361299B CN 103361299 B CN103361299 B CN 103361299B CN 201210096966 A CN201210096966 A CN 201210096966A CN 103361299 B CN103361299 B CN 103361299B
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Abstract
The present invention relates to method and the test kit of separate stem cells.Wherein, the present invention proposes a kind of composition for separating of stem cell, it comprises: Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in described balanced salt solution.Utilize said composition, can effective separate stem cells.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method and a kit for separating stem cells, and more particularly relates to application of iohexol in preparing a stem cell purification solution, a composition for separating stem cells, a method for separating stem cells, a kit for separating stem cells and application of the kit in separating stem cells.
Background
Human bone marrow and umbilical cord blood contain a large number of hematopoietic cells as well as various types of stem cells and a large number of mature cells. The stem cells of each line and the mature leukocytes are nucleated cells. The nucleated cells include various stem cells and mono/polynuclear cells, as well as lymphocytes and mesenchymal cells.
The method for separating the erythroid cells and the nucleated cells of the in vitro bone marrow comprises a machine centrifugation method and a sedimentation method. The machine centrifugation method is widely used abroad due to its fully closed operation and stable and safe separation effect. But the equipment is expensive, so the popularization is difficult at home at present. There are also a number of methods: magnetic bead method, immunofluorescence labeling method, flow cytometry method and the like. The problems common to these methods are: 1) the obtained stem cells are not pure, 2) the use is inconvenient, the cost is high, and 3) the technical equipment requirement is high, and the operation has high requirement on technical personnel.
Thus, the current techniques for isolating stem cells remain to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. To this end, it is an object of the present invention to propose a means that enables efficient isolation of stem cells.
The present invention has been completed based on the following findings of the inventors: iohexol is readily soluble in water and buffers, up to 80% by weight, and the iohexol solution can be autoclaved, is non-toxic, has low viscosity, and does not affect cell osmotic pressure. The inventors have surprisingly found that iohexol, when applied to a purification solution as a density gradient centrifugation medium, can be widely applied to the purification of various types of cells, subcellular organelles and macromolecular complexes with a significantly better efficiency than Percoll.
To this end, in a first aspect of the invention, the invention proposes the use of iohexol in the preparation of a stem cell purification solution. By applying iohexol to the preparation of the stem cell purification solution, the efficiency of separating stem cells can be effectively improved.
In a second aspect of the invention, the invention features a composition for isolating stem cells. According to an embodiment of the invention, the composition comprises: iohexol; and a balanced salt solution, wherein the iohexol is dissolved in the balanced salt solution. The composition is used as a stem cell purification solution, and the efficiency of separating stem cells from a biological sample containing the stem cells can be effectively improved.
In a third aspect of the invention, the invention features a method of isolating a stem cell. According to an embodiment of the invention, the method comprises the following: mixing a biological sample containing stem cells with a red blood cell separation solution, and standing and settling to obtain an upper cell solution containing the stem cells; centrifuging and concentrating the upper layer cell sap containing the stem cells to obtain cell sediment; resuspending the cell pellet with a first stem cell purification solution to obtain a suspension; adding the suspension into a second stem cell purification solution, centrifuging, and collecting stem cells, wherein the red blood cell separation solution is a 0.2-0.7 wt% methyl cellulose aqueous solution, the first stem cell purification solution is a balanced salt solution, and the second stem cell purification solution is the composition for separating stem cells (namely containing iohexol; and the balanced salt solution, wherein the iohexol is dissolved in the balanced salt solution). The inventors have found that the efficiency of stem cell isolation can be effectively improved by using the above-described method for stem cell isolation.
In a fourth aspect of the invention, the invention provides a kit for isolating stem cells. According to an embodiment of the invention, the kit comprises: separating liquid of red blood cells; a first stem cell purification solution; and a second stem cell purification solution, wherein the red blood cell separation solution is a 0.2-0.7 wt% methylcellulose aqueous solution, preferably a 0.5 wt% methylcellulose aqueous solution, the first stem cell purification solution is a balanced salt solution and the second stem cell purification solution is a composition of the aforementioned separated stem cells (i.e., comprising iohexol; and a balanced salt solution in which iohexol is dissolved). The method for isolating stem cells can be efficiently carried out using the kit, and thus, the efficiency of isolating stem cells can be effectively improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
fig. 1 is a schematic flow chart showing a method of isolating stem cells according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
The present invention has been completed based on the following findings of the inventors: the existing gradient centrifugation reagents such as Percoll and Filcoll solutions are inefficient when applied to the purification of nucleated cells, especially stem cells. Because the time of the cells in vitro is long, and the separation process is influenced by temperature and reagents, the recovery rate of the nucleated cells obtained by the prior art can only be maintained at 80-90%, the residual rate of the red blood cells can reach about 4%, and because the time of the operation in vitro exceeds 2 hours, the differentiation pluripotency of the obtained stem cells can be greatly influenced, and the differentiation potential of the stem cells is reduced. Iohexol (Iohexol, chemical name: triiodotriaminohexaol benzene, trade name: nyodenz, CAS No.66108-95-0) is a compound with a molecular weight of 821, a density of 2.1g/ml, which is non-ionic and non-toxic to cells. The inventors found that iohexol can be effectively applied to separation or purification of various biological substances such as cells, organelles, biological macromolecules and viruses, and that iohexol is stable at high temperature and high pressure, so that a solution prepared from iohexol can be sterilized at high temperature and high pressure, and iohexol is suitable as a gradient centrifugation reagent for separating stem cells.
In a first aspect of the invention, the invention proposes the use of iohexol in the preparation of a stem cell purification solution. The stem cell purification solution prepared by using iohexol can be effectively used for separating and purifying stem cells, and is particularly suitable for separating and purifying stem cells from spinal fluid and umbilical cord blood, preferably anticoagulant spinal fluid and umbilical cord blood.
In a second aspect of the invention, the invention features a composition for isolating stem cells. According to an embodiment of the present invention, the composition for isolating stem cells is a solution of iohexol in a balanced salt solution, i.e. the composition for isolating stem cells comprises iohexol and a balanced salt solution, wherein said iohexol is dissolved in said balanced salt solution. The composition can be used as a gradient centrifugation reagent to effectively separate stem cells from a sample containing the stem cells by gradient centrifugation.
According to the embodiment of the present invention, the type of the balanced salt solution is not particularly limited as long as it can maintain the osmotic pressure balance of the cells. According to one embodiment of the present invention, the Balanced Salt Solution that may be used is at least one of HBSS (Hank 'S Balanced Salt Solution) and GBSS (GEY' S Balanced Salt Solution). The preferred balanced salt solution is HBSS. According to an embodiment of the invention, the composition has a pH of 7 to 8. Thus, stem cells can be isolated more efficiently.
According to the embodiment of the present invention, the concentration of iohexol is not particularly limited. According to one embodiment of the invention, the balanced salt solution contains 8-15g iohexol, preferably 13g iohexol per 100ml balanced salt solution. This can further improve the efficiency of stem cell separation by gradient centrifugation. Compositions containing iohexol in the balanced salt according to embodiments of the invention may conveniently be obtained, for example, by mixing a quantity of iohexol, for example 13 grams iohexol, with 100ml of HBSS solution (commercially available, for example from Invitrogen) and stirring for a period of time, for example for more than 1 hour, and then storing at 4 ℃ in the dark until ready for use.
In addition, according to an embodiment of the present invention, the composition may further comprise a cell growth factor, preferably further comprise 10 to 30ng/ml of human basic fibroblast growth factor and 10 to 30ng/ml of human stem cell growth factor-alpha. Thus, the inventors found that the in vitro survival rate of stem cells can be effectively improved and the differentiation potential of the stem cells can be maintained by adding growth factors, especially stem cell growth factors, to the composition.
In a third aspect of the invention, the invention features a method of isolating a stem cell. Referring to fig. 1, the method of isolating stem cells includes the steps of:
first, red blood cells are isolated. According to the embodiment of the invention, the biological sample containing the stem cells can be mixed with the erythrocyte separating solution and placed still for sedimentation, so that the lower layer of the erythrocyte sedimentation solution is formed, and the upper layer of the cell solution containing the stem cells is obtained. The type of the term "erythrocyte separating medium" used herein is not particularly limited as long as erythrocytes can be sufficiently separated from nucleated cells including stem cells by sedimentation. According to one embodiment of the invention, the red blood cell separation solution used is a 0.2-0.7 wt% aqueous solution of methylcellulose, preferably a 0.5 wt% aqueous solution of methylcellulose. The settling time is not particularly limited according to the embodiment of the present invention, and settling may be performed for 45 to 60 minutes according to the embodiment of the present invention. Thus, erythrocytes can be efficiently and sufficiently separated from nucleated cells including stem cells. Thereby obtaining the upper layer cell sap containing the stem cells. According to an embodiment of the present invention, the type of the stem cell-containing biological sample is not particularly limited, and may be any stem cell-containing biological sample isolated from any part of a living body such as a human body. According to an embodiment of the present invention, the biological sample containing stem cells may be at least one selected from spinal fluid and cord blood, preferably anticoagulated spinal fluid or cord blood. Thereby, a large number of stem cells, in particular mesenchymal stem cells, may be obtained. According to the embodiment of the present invention, the mixing ratio of the stem cell-containing biological sample and the red blood cell separation solution is not particularly limited. According to one embodiment of the invention, the mixing ratio is chosen such that the final mixed liquor contains 0.1 wt% of methylcellulose. For example, in the case of anticoagulated spinal fluid or umbilical cord blood, the anticoagulated spinal fluid or umbilical cord blood is mixed with a 0.5% by weight aqueous solution of methyl cellulose (red blood cell separation solution) at a volume ratio of 4: 1. This can further improve the efficiency of separating red blood cells, and thus can further improve the efficiency of finally separating stem cells. According to the embodiment of the present invention, the operation of removing red blood cells by sedimentation can be repeated for the separated supernatant-containing cell liquid to further remove the remaining red blood cells.
Next, the upper cell sap was collected, and the upper cell sap containing the stem cells was concentrated by centrifugation, thereby obtaining a cell pellet. And then resuspending the resulting cell pellet using a first stem cell purification solution. It should be noted that the terms "first" and "second" are used herein for distinguishing between them and not for distinguishing between them in any way whether or not they are sequential or important. Herein, the first stem cell purification solution used is at least one selected from the group consisting of a balanced salt solution and a cell culture solution. According to an embodiment of the present invention, the type of the balanced salt solution and the cell culture solution is not particularly limited, and examples of the balanced salt solution that can be used include, but are not limited to, GBSS solution (sometimes also referred to as GBSS directly), HBSS solution (sometimes also referred to as HBSS directly), preferably the balanced salt solution that can be used is HBSS, and the culture solution that can be used is MEM culture solution. According to the embodiment of the present invention, the conditions for centrifugal concentration of the upper cell sap containing the stem cells are not particularly limited. According to one embodiment of the invention, centrifugation at 1200rpm for 5 minutes may be used to achieve effective concentration of the cells. The resulting cell pellet can be resuspended in an amount of balanced salt solution, e.g., 10ml of balanced salt solution (first stem cell purification solution), to obtain a suspension containing stem cells.
Next, after obtaining a suspension containing stem cells, the obtained suspension may be added to a second cell purification solution to perform gradient centrifugation to separate the stem cells. According to an embodiment of the present invention, the second stem cell purification solution is the composition described above, i.e., a solution of iohexol in a balanced salt solution (in other words, the composition for separating stem cells comprises iohexol and a balanced salt solution in which the iohexol is dissolved). Thus, nucleated cells can be efficiently separated from other cells by gradient centrifugation, thereby facilitating the separation of stem cells, particularly mesenchymal stem cells (typically located in the middle of the centrate). According to the embodiment of the present invention, the conditions of centrifugation are not particularly limited. According to an embodiment of the present invention, gradient centrifugation at 4 ℃ for 22 minutes may be used. Thus, stem cells can be isolated more efficiently. With respect to the second stem cell-purifying solution, all the technical features and advantages described above for the composition for isolating stem cells can be applied thereto, and for convenience, they will not be described in detail. The conditions for performing gradient centrifugation using the second stem cell-purified solution are not particularly limited.
According to the embodiment of the present invention, after obtaining stem cells, the obtained stem cells may be washed with a cell treatment solution. According to an embodiment of the present invention, the cell treatment liquid that may be used is at least one of physiological saline and phosphate buffer. According to an embodiment of the present invention, the obtained stem cells may be washed at least twice using the cell treatment solution, whereby the purity of the obtained stem cells can be ensured. According to an embodiment of the present invention, the type of the cell treatment solution is not particularly limited, and may be at least one selected from a phosphate buffer solution and a physiological saline. This can further improve the efficiency of washing stem cells. The expression "at least one selected from the group consisting of phosphate buffer and physiological saline" as used herein means that the phosphate buffer may be used alone, the physiological saline may be used alone, or a combination of the phosphate buffer and the physiological saline may be used. The "combination of phosphate and physiological saline" used herein should be broadly understood, and may be a mixture of phosphate buffer and physiological saline for washing the stem cells, or a mixture of phosphate buffer and physiological saline for washing the stem cells, wherein if the phosphate buffer and physiological saline are used for washing the stem cells, respectively, the sequence is not particularly limited, and the phosphate buffer may be used first, followed by the physiological saline, or the physiological saline may be used first, followed by the phosphate buffer.
According to an embodiment of the present invention, at least one of the red blood cell separation solution, the first stem cell purification solution, the second stem cell purification solution, and the cell processing solution used in the present invention may further include: 10-30ng/ml human basic fibroblast growth factor and 10-30ng/ml human stem cell growth factor-alpha. Preferably, the red blood cell separation solution, the first stem cell purification solution, the second stem cell purification solution and the cell treatment solution further comprise: 10-30ng/ml human basic fibroblast growth factor and 10-30ng/ml human stem cell growth factor-alpha. Thus, the inventors found that the in vitro survival rate of stem cells can be effectively improved and the differentiation potential of the stem cells can be maintained by adding growth factors, particularly stem cell growth factors.
By using the method for separating the stem cells, the stem cells in the spinal cord fluid and the umbilical cord blood can be separated, the survival rate of the separated stem cells reaches more than 99 percent, and the yield of the stem cells reaches 90 percent. According to the embodiment of the invention, the operations can be operated in the biosafety cabinet according to the operating rules, and the separation cannot be polluted by the reagent. Meanwhile, the surfaces of the stem cells separated by the method according to the embodiment of the invention are free from any markers, and the biological activity of the cells is not changed. In addition, according to the method of the embodiment of the invention, because the growth factor is added to the reagent in the separation process, the survival rate of the stem cells is improved in the separation process, and the pluripotency of the stem cells is maintained.
In a fourth aspect of the invention, the invention provides a kit for isolating stem cells. According to an embodiment of the invention, the kit comprises: the kit comprises a red blood cell separating solution, a first stem cell purifying solution and a second stem cell purifying solution. According to an embodiment of the present invention, the kit may further include a cell processing solution.
According to the embodiment of the present invention, the red blood cell separation solution is a 0.2-0.7 wt% methylcellulose aqueous solution, preferably a 0.5 wt% methylcellulose aqueous solution, which can be effectively used for removing red blood cells by sedimentation separation. According to an embodiment of the present invention, the first stem cell purification solution is at least one selected from the group consisting of a balanced salt solution and a cell culture solution. According to an embodiment of the present invention, the types of the balanced Salt Solution and the cell culture Solution are not particularly limited, and according to an embodiment of the present invention, at least one of HBSS (Hank 'S balanced Salt Solution) and GBSS (GEY' S balanced Salt Solution) may be used. A preferred balanced salt solution is HBSS, and the culture medium that can be used is MEM culture medium. The centrifugally concentrated cell pellet containing the stem cells can be efficiently resuspended for subsequent processing. According to an embodiment of the present invention, the second stem cell purification solution is the composition described above, i.e., a solution of iohexol in a balanced salt solution (in other words, the composition for separating stem cells comprises iohexol and a balanced salt solution in which the iohexol is dissolved). Thus, nucleated cells can be efficiently separated from other cells by gradient centrifugation, thereby facilitating the separation of stem cells, particularly mesenchymal stem cells (typically located in the middle of the centrate). With respect to the second stem cell-purifying solution, all the technical features and advantages described above for the composition for isolating stem cells can be applied thereto, and for convenience, they will not be described in detail. According to an embodiment of the present invention, the cell treatment solution is at least one of a phosphate buffer and a physiological saline. Thus, the stem cells separated can be easily washed with the cell treatment solution. Thus, the method for isolating stem cells can be efficiently carried out using the kit, and stem cells can be efficiently isolated from a biological sample containing stem cells.
According to an embodiment of the present invention, at least one of the red blood cell separation solution, the first stem cell purification solution, the second stem cell purification solution, and the cell processing solution used in the present invention may further include: 10-30ng/ml human basic fibroblast growth factor and 10-30ng/ml human stem cell growth factor-alpha. Preferably, the red blood cell separation solution, the first stem cell purification solution, the second stem cell purification solution and the cell treatment solution further comprise: 10-30ng/ml human basic fibroblast growth factor and 10-30ng/ml human stem cell growth factor-alpha. Thus, the inventors found that the in vitro survival rate of stem cells can be effectively improved and the differentiation potential of the stem cells can be maintained by adding growth factors, particularly stem cell growth factors. According to an embodiment of the present invention, the red blood cell separation solution, the first stem cell purification solution and the second stem cell purification solution are respectively disposed in different containers. Therefore, the kit can be conveniently used for separating stem cells. The kit can be conveniently produced industrially, and the mesenchymal stem cells with high quality and high purity can be conveniently obtained by using the kit for scientific research or clinical treatment.
Thus, in a fifth aspect of the invention, the invention proposes the use of the above-described kit for isolating stem cells. By adopting the kit of the embodiment of the invention, the method for separating the stem cells can be effectively implemented. All features and advantages relating to the separation method are suitable here and will not be described again for the sake of simplicity.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be performed according to the third edition of the molecular cloning, laboratory Manual, or related products, and the reagents and products used are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.
Example 1 (materials in general and methods in general)
The kit comprises:
reagent A: erythrocyte separating medium
0.5-0.6% of Methylcellulose (MC) (FLUN K, Switzerland).
The average viscosity of methylcellulose is 4,000cP, and pre-cooled 4 ℃ physiological saline is firstly used for preparing 0.4-0.6 wt% methylcellulose solution, the solution is placed at 4 ℃ overnight, and then the solution is repeatedly shaken up until the methylcellulose is completely dissolved. The resulting methylcellulose solution was autoclaved at 0.1MPa for 15 minutes, cooled to 4 ℃ and then vigorously shaken. The methylcellulose is colorless and transparent after being completely dissolved, has pH of 7-8, and is stored at 4 deg.C for use.
And (3) reagent B: stem cell purification solution I
HBSS solution (available from Invitrogen corporation), GBSS solution or MEM culture solution
And (3) reagent C: stem cell purification solution II
8-15 wt% iohexol solution: a predetermined amount (8-15g) of iohexol (Nycodenz) was mixed with 100ml of HBSS solution (Invitrogen) and stirred for 1 hour or more, and finally stored at 4 ℃ in the dark. The pH value is 7-8.
Iohexol (Nycodenz) is a white and highly hydrophilic powder, the chemical component of which is Iohexol (CAS No.66108-95-0), and can be prepared into a totipotent non-ionic density gradient separation liquid.
Iohexol (Nycodenz) has a molecular weight of 821 and a density of 2.1g/ml, is a non-ionic compound which is non-toxic to cells, and can be used for separating or purifying various biological substances such as cells, organelles, biological macromolecules, viruses and the like. The separated liquid prepared from iohexol (Nycodenz) can be sterilized at high temperature and high pressure.
And (3) reagent D: cell treatment liquid
Phosphate buffer or physiological saline
After the reagent is prepared, the endotoxin content is tested to be less than or equal to 0.5EU/ml after high-temperature sterilization at 121 ℃ for 1 hour. Are respectively stored in the reagent kit. Growth factors were added prior to use, and human basic fibroblast growth factor (hFGF2) was added to each reagent at a final concentration of 10-30ng/ml, as well as human stem cell growth factor-alpha (pro-stem cell growth factor) at a final concentration of 10-30 ng/ml.
General procedure
I. Separating red blood cells:
the method comprises the following steps: mixing collected anticoagulant marrow fluid or umbilical cord blood with erythrocyte separating medium, placing in 100ml open infusion apparatus, suspending and standing in ultra-clean bench at room temperature, settling for 45-60 min, discharging erythrocyte in lower layer, collecting upper layer cell fluid, and centrifuging and concentrating. And (4) resuspending, centrifuging and concentrating the obtained cell precipitate by using the stem cell purification solution I.
The method 2 comprises the following steps: mixing collected anticoagulant marrow fluid or umbilical cord blood with erythrocyte separating medium, adding sample into erythrocyte separating medium container according to predetermined ratio, standing in superclean bench at room temperature, settling for 45-60 min, collecting upper layer cell fluid, and centrifuging and concentrating. And (4) resuspending, centrifuging and concentrating the obtained cell precipitate by using the stem cell purification solution I.
II. Gradient centrifugation for stem cells:
and (3) slowly adding a suspension obtained by resuspending, centrifuging and concentrating the cell sediment by using the cell purification solution I to the stem cell purification solution II, and slightly dropwise adding to avoid disturbing the boundary layer. Then, the centrifuge tube was centrifuged for 22 minutes, and a middle milky white stem cell layer was collected, and the cells were repeatedly washed 2 times with a cell treatment solution to obtain stem cells.
Example 2
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
HBSS solution (purchased from Invitrogen) was supplemented with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
13% Nycodenz solution: nycodenz 13g, HBSS solution (Invitrogen) 100ml, stirred for > 1 hour, and added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor) were added to the phosphate buffer.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium were mixed uniformly at a ratio of 4: 1 (v: v) to give a final concentration of 0.1 wt% (methyl cellulose, the same applies hereinafter) of the erythrocyte separating medium, and placed in a 100ml open infusion set. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, completely discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below an infusion apparatus, settling for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm5 minutes, and concentrating. Diluting the cells with 10ml of reagent B, and buffering the collected cell suspensionAdd slowly to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, and detecting the cell survival rate by using bromophenol blue, wherein the cell survival rate reaches 99%. Taking 5 microliter stem cell sample, counting cells, and obtaining at least 4 × 10 cells per 80ml bone marrow or umbilical cord blood7A stem cell
Example 3
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
HBSS solution (purchased from Invitrogen) was supplemented with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
13% Nycodenz solution: nycodenz 13g, HBSS solution (Invitrogen) 100ml, stirred for > 1 hour, and added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
Human basic fibroblast growth factor (hFGF2) 20ng/ml and human stem cell growth factor-alpha (protostem cell growth factor) 20ng/ml were added to the saline.
And (3) placing the collected 80ml of anticoagulated bone marrow liquid or umbilical cord blood and 20ml of reagent A in a bottle, standing and settling for 45-60 minutes in a clean bench at room temperature, completely precipitating erythrocytes at the lower layer, collecting cell sap at the upper layer, and centrifuging and concentrating at 1200rpm5 minutes. The precipitated fine was diluted with 10ml of reagent BClumping, the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with reagent D for 2 times. The cells were diluted with 5ml of reagent D for use. Taking 5 microliters, detecting that the cell survival rate reaches 99% by using bromophenol blue, and then taking 5 microliters for cell counting. The conclusion is that every 80ml bone marrow or umbilical cord blood is separated to obtain 4X 107And (4) stem cells.
Example 4
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
GBSS solution (purchased from Invitrogen) was supplemented with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
13% Nycodenz solution: nycodenz 13g, GBSS solution (Invitrogen) 100ml, stirred for > 1 hour, and added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor) were added to the phosphate buffer.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium are mixed uniformly according to a ratio of 4: 1 (v: v) to make the final concentration of the erythrocyte separating medium be 0.1 wt%, and the mixture is placed in a 100ml open infusion apparatus. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, and completely standingAnd (3) discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below the infusion apparatus, descending for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm for 5 minutes, and concentrating. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 98% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
Example 5
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
GBSS solution (purchased from Invitrogen) was supplemented with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
13% Nycodenz solution: nycodenz 13g, GBSS solution (Invitrogen) 100ml, stirred for > 1 hour, and added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
Human basic fibroblast growth factor (hFGF2) 20ng/ml and human stem cell growth factor-alpha (protostem cell growth factor) 20ng/ml were added to the saline.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium are mixed uniformly according to a ratio of 4: 1 (v: v) to make the final concentration of the erythrocyte separating medium be 0.1 wt%, and the mixture is placed in a 100ml open infusion apparatus. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, completely discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below an infusion apparatus, settling for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm5 minutes, and concentrating. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 98% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
Example 6
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
To MEM culture medium (purchased from Invitrogen) were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
13% Nycodenz solution: nycodenz 13g, HBSS solution (Invitrogen) 100ml, stirred for > 1 hour, and added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor) were added to the phosphate buffer.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium are mixed uniformly according to a ratio of 4: 1 (v: v) to make the final concentration of the erythrocyte separating medium be 0.1 wt%, and the mixture is placed in a 100ml open infusion apparatus. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, completely discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below an infusion apparatus, settling for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm5 minutes, and concentrating. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 99% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
Example 7
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
To MEM culture medium (purchased from Invitrogen) were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
13% Nycodenz solution: nycodenz 13g, HBSS solution (Invitrogen) 100ml, stirred for > 1 hour, and added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
Human basic fibroblast growth factor (hFGF2) 20ng/ml and human stem cell growth factor-alpha (protostem cell growth factor) 20ng/ml were added to the saline.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium are mixed uniformly according to a ratio of 4: 1 (v: v) to make the final concentration of the erythrocyte separating medium be 0.1 wt%, and the mixture is placed in a 100ml open infusion apparatus. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, completely discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below an infusion apparatus, settling for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm5 minutes, and concentrating. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 99% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
Example 8
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
To MEM culture medium (purchased from Invitrogen) were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
8% Nycodenz solution: nycodenz 8g, HBSS solution (Invitrogen) 100ml, stirred for > 1 hour, and added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
Human basic fibroblast growth factor (hFGF2) 20ng/ml and human stem cell growth factor-alpha (protostem cell growth factor) 20ng/ml were added to the saline.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium are mixed uniformly according to a ratio of 4: 1 (v: v) to make the final concentration of the erythrocyte separating medium be 0.1 wt%, and the mixture is placed in a 100ml open infusion apparatus. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, completely discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below an infusion apparatus, settling for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm for 5 minutes, and concentrating. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 99% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
Example 9
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
To MEM culture medium (purchased from Invitrogen) were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
11% Nycodenz solution: nycodenz 11g, HBSS solution (Invitrogen) 100ml, stirred for > 1 hour, added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
Human basic fibroblast growth factor (hFGF2) 20ng/ml and human stem cell growth factor-alpha (protostem cell growth factor) 20ng/ml were added to the saline.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium are mixed uniformly according to a ratio of 4: 1 (v: v) to make the final concentration of the erythrocyte separating medium be 0.1 wt%, and the mixture is placed in a 100ml open infusion apparatus. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, completely discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below an infusion apparatus, settling for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm5 minutes, and concentrating. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for further use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 99% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
Example 10
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
To MEM culture medium (purchased from Invitrogen) were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent C: stem cell purification solution II
15% Nycodenz solution: nycodenz 15g, HBSS solution (Invitrogen) 100ml, stirred for > 1 hour, added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent D: cell treatment liquid
Human basic fibroblast growth factor (hFGF2) 20ng/ml and human stem cell growth factor-alpha (protostem cell growth factor) 20ng/ml were added to the saline.
The collected 80ml of anticoagulated bone marrow fluid or umbilical cord blood and 20ml of 0.5 wt% erythrocyte separating medium are mixed uniformly according to a ratio of 4: 1 (v: v) to make the final concentration of the erythrocyte separating medium be 0.1 wt%, and the mixture is placed in a 100ml open infusion apparatus. Suspending and standing in a superclean bench at room temperature, settling for 45-60 min, discharging the red blood cells at the lower layer, allowing the boundary liquid level of red blood cells and white blood cells to be at the upper end of a semi-transparent silica gel infusion tube with diameter of 5mm connected below the infusion apparatus, settling for 5 min, and slowly dischargingThe lower layer of red blood cells are collected, and the upper layer of cell sap is centrifuged and concentrated at 1200rpm for 5 minutes. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 99% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
Example 11
Reagent A: erythrocyte separating medium
To 0.5% methylcellulose were added 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor).
And (3) reagent B: stem cell purification solution I
HBSS (purchased from Invitrogen) was supplemented with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor), and phenol red was added.
And (3) reagent C: stem cell purification solution II
13% Nycodenz solution: nycodenz 13g, 100ml HBSS solution (invitrogen), stirred for > 1 hour, added with 20ng/ml human basic fibroblast growth factor (hFGF2) and 20ng/ml human stem cell growth factor-alpha (protostem cell growth factor), and phenol red.
And (3) reagent D: cell treatment liquid
Human basic fibroblast growth factor (hFGF2) 20ng/ml and human stem cell growth factor-alpha (protostem cell growth factor) 20ng/ml were added to the saline.
Mixing 80ml of anticoagulated marrow fluid or umbilical cord blood with 20ml of 0.5 wt% erythrocyte separating medium at ratio of 4: 1 (v: v)The mixture was homogenized until the final concentration of the red blood cell separation solution was 0.1 wt%, and placed in a 100ml open infusion set. Suspending and standing in a superclean workbench at room temperature, settling for 45-60 minutes, completely discharging the lower layer of red blood cells, enabling the boundary liquid level of the red blood cells and the white blood cells to be positioned at the upper end of a semitransparent silica gel infusion tube with the diameter of 5mm connected below an infusion apparatus, settling for 5 minutes, slowly discharging the lower layer of red blood cells, collecting the upper layer of cell sap, centrifuging at 1200rpm5 minutes, and concentrating. The cells were diluted with 10ml of reagent B and the collected cell suspension was slowly added to reagent C. 900g, gradient centrifugation at 4 ℃ for 22 minutes, collection of a middle milky white stem cell layer, and repeated washing of cells with a cell treatment solution for 2 times. Finally, the cells were diluted with 5ml of the cell treatment solution for use. Taking 5 microliters of stem cell samples, detecting that the cell survival rate reaches 99% by using bromophenol blue, then taking 5 microliters of stem cell samples, counting the cells, and concluding that every 80ml of bone marrow or umbilical cord blood is separated to obtain 4 multiplied by 107And (4) stem cells.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A method of isolating stem cells, comprising the steps of:
mixing a biological sample containing stem cells with a red blood cell separation solution, and standing and settling to obtain an upper cell solution containing the stem cells;
centrifuging and concentrating the upper layer cell sap containing the stem cells to obtain cell sediment;
resuspending the cell pellet with a first stem cell purification solution to obtain a suspension;
adding the suspension to a second stem cell purification solution and centrifuging to collect stem cells,
wherein,
the erythrocyte separating medium is 0.2-0.7 wt% methyl cellulose water solution,
the first stem cell purification solution is at least one selected from a balanced salt solution and a cell culture solution,
the second stem cell purification solution comprises:
iohexol; balancing the salt solution; 20ng/ml human basic fibroblast growth factor and 20ng/ml human stem cell growth factor-alpha,
wherein,
the iohexol is dissolved in the balanced salt solution, each 100ml of balanced salt solution contains 8-15g of iohexol,
the pH value of the second stem cell purification solution is 7-8.
2. The method of claim 1, wherein said balanced salt solution contains 13g iohexol per 100 ml.
3. The method of claim 1, wherein the red blood cell separation fluid is a 0.5 wt% aqueous solution of methylcellulose.
4. The method according to claim 1, wherein the balanced salt solution is at least one selected from the group consisting of HBSS and GBSS, and the cell culture solution is MEM culture solution.
5. The method of claim 4, wherein the balanced salt solution is HBSS.
6. The method of claim 1, wherein the biological sample containing stem cells is at least one selected from the group consisting of spinal fluid and cord blood.
7. The method of claim 6, wherein the biological sample containing stem cells is anticoagulated spinal fluid or umbilical cord blood.
8. The method of claim 6, wherein the stem cell-containing biological sample and the red blood cell centrate are mixed in a volume ratio of 4: 1 and mixing.
9. The method of claim 6, further comprising washing the obtained stem cells with a cell treatment solution at least once,
wherein,
the cell treatment solution is at least one selected from the group consisting of a phosphate buffer solution and a physiological saline.
10. The method according to claim 9, wherein the obtained stem cells are washed twice with the cell treatment solution.
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