CN113046317A - Method for extracting platelet-rich plasma based on preparation of umbilical cord blood hematopoietic stem cells - Google Patents
Method for extracting platelet-rich plasma based on preparation of umbilical cord blood hematopoietic stem cells Download PDFInfo
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- CN113046317A CN113046317A CN202110349072.7A CN202110349072A CN113046317A CN 113046317 A CN113046317 A CN 113046317A CN 202110349072 A CN202110349072 A CN 202110349072A CN 113046317 A CN113046317 A CN 113046317A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0644—Platelets; Megakaryocytes
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a method for extracting platelet-rich plasma on the basis of preparation of umbilical cord blood hematopoietic stem cells, which comprises the steps of specimen pretreatment, red blood cell removal, umbilical cord blood plasma collection, platelet-rich plasma preparation, platelet-rich plasma identification and hematopoietic stem cell identification. The invention has the beneficial effects that: on the basis of not influencing the preparation and extraction of umbilical cord blood hematopoietic stem cells, platelets in plasma are concentrated and separated to obtain platelet-rich plasma, the identification of the extracted hematopoietic stem cells and platelet-rich plasma does not influence the extraction quality of the hematopoietic stem cells, and the extraction rate of the platelet-rich plasma reaches 60%.
Description
Technical Field
The invention relates to a method for extracting platelet-rich plasma, in particular to a method for extracting platelet-rich plasma on the basis of preparation of umbilical cord blood hematopoietic stem cells, and belongs to the technical field of biology.
Background
Platelet-rich plasma (PRP) is plasma containing high concentrations of platelets isolated by ex vivo treatment of autologous peripheral blood, with different centrifugal forces. PRP is derived from platelets and contains various abundant growth factors and proteins, such as platelet-derived growth factors, transforming growth factor beta, vascular endothelial cell growth factors, epidermal growth factors, insulin-like growth factors and the like; these growth factors play important roles in various aspects such as promotion of cell proliferation, redifferentiation, chemotaxis, and stimulation of angiogenesis and repair.
Cord blood is blood that remains in the placenta and umbilical cord after the fetus is delivered, ligated and severed, and is typically disposed of as medical waste. Research in recent ten years shows that cord blood contains hematopoietic stem cells which can restore human hematopoietic and immune systems, can be used for hematopoietic stem cell transplantation to treat more than 80 diseases, and particularly, the cord blood is more and more regarded by the nation as a very important human biological resource. The method for extracting the platelet-rich plasma on the basis of the preparation of the umbilical cord blood hematopoietic stem cells is provided based on the application.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for extracting platelet-rich plasma from plasma obtained by extracting hematopoietic stem cells from umbilical cord blood, which comprises separating platelet-rich plasma from the plasma obtained by extracting hematopoietic stem cells from umbilical cord blood. The obtained umbilical cord blood source platelet-rich plasma is subjected to detection of a platelet surface marker (CD 41 a) by flow cytometry, the platelets are counted by a hemocytometer, the concentration of the platelets obtained by a double platform is more than 3 times that of original blood, and the extraction rate is more than 60%.
The invention realizes the purpose through the following technical scheme: a method for extracting platelet-rich plasma based on cord blood hematopoietic stem cell preparation comprises the following steps:
firstly, pretreating a specimen, and disinfecting the surface of the specimen to remove surface stains and possible bacteria from the specimen to enter blood;
removing red blood cells, adding hydroxyethyl starch into the umbilical cord blood specimen, wherein the hydroxyethyl starch (HES) can be combined with negative charges on erythrocyte membranes to enable concave surfaces of the red blood cells to be attached to increase sedimentation speed, and removing the red blood cells by utilizing the difference of sedimentation speeds of the red blood cells and white blood cells;
step three, collecting umbilical cord blood plasma, centrifuging the transfer bag from which the red blood cells are removed in the step three again, transferring the supernatant into a new bag, namely collecting the plasma; freezing and storing the hematopoietic stem cells according to the umbilical cord blood freezing and storing process rule;
step four, preparing platelet-rich plasma, centrifuging the plasma obtained in the step above to remove red blood cells, collecting supernate, centrifuging again, and taking precipitate to obtain the platelet-rich plasma of the umbilical cord blood;
and step five, identifying the platelet-rich plasma and hematopoietic stem cells, and carrying out flow detection and counting on the surface markers of the platelet-rich plasma obtained in the step four.
As a still further scheme of the invention: in the second step, the cord blood specimen is prepared according to the volume of cord blood: hydroxyethyl starch = 5: 1 the desired amount of hydroxyethyl starch is added. Centrifuging at 500rpm for 7min, and separating into three layers, upper layer plasma, middle layer stem cells, and lower layer red blood cells. The hemostatic clamp of the 150ml transfer bag was clamped and the stem cells and plasma were slowly squeezed into another 250ml transfer bag.
As a still further scheme of the invention: in the third step, the 250ml transfer bag in the upper step is centrifuged at 1800rpm for 5min and divided into three clear layers, namely upper plasma layer, middle stem cells and a little red blood cells in the lower layer. The hemostatic clamp of the 150ml transfer bag was opened and the plasma in the upper layer of the 250ml transfer bag was slowly expressed and drained into the 150ml transfer bag.
As a still further scheme of the invention: in step four, the collected plasma was centrifuged for 1500prm 5min, this step again removing red blood cells; the collected plasma was centrifuged again at 2500prm for 10 min. Discarding the upper layer of yellow clear supernatant with a length of 2-3cm, and the lower layer is platelet-rich plasma.
As a still further scheme of the invention: and in the fifth step, the separated platelet-rich plasma and hematopoietic stem cells of the umbilical cord blood are subjected to cell viability, full-automatic blood cell counter analysis and flow cytometry analysis, and are used for identifying the platelet-rich plasma and the hematopoietic stem cells of the umbilical cord blood.
The invention has the beneficial effects that: the method for extracting the platelet-rich plasma based on the preparation of the umbilical cord blood hematopoietic stem cells is reasonable in design, can obtain the platelet-rich plasma which is more than 3 times of the whole umbilical cord blood by utilizing a biotechnology means, contains high-concentration growth factors, and can replace a supplement reagent fetal bovine serum in the traditional mesenchymal stem cell culture. The platelet-rich plasma has the following advantages:
1) the method has the advantages of rich raw materials, simple operation method and less equipment requirement;
2) the immunogenicity of the umbilical cord blood source platelet rich plasma is low;
3) the umbilical cord blood platelet-rich plasma contains various high-concentration growth factors, and can support osteogenic differentiation, adipogenic differentiation and growth of induced pluripotent stem cells of mesenchymal stem cells;
4) after the extraction of the umbilical cord blood hematopoietic stem cells is finished, on the basis of not changing the original preparation process, platelet-rich plasma is further extracted from the collected plasma, so that the surplus materials are reused, the cord blood genetic resource is reused, and two biological products can be obtained from one raw material.
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FIG. 1 is a schematic flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
Referring to fig. 1, a method for extracting platelet-rich plasma based on preparation of cord blood hematopoietic stem cells comprises the following steps:
firstly, pretreating a specimen, and disinfecting the surface of the specimen to remove surface stains and possible bacteria from the specimen to enter blood;
removing red blood cells, adding hydroxyethyl starch into the umbilical cord blood specimen, wherein the hydroxyethyl starch (HES) can be combined with negative charges on erythrocyte membranes to enable concave surfaces of the red blood cells to be attached to increase sedimentation speed, and removing the red blood cells by utilizing the difference of sedimentation speeds of the red blood cells and white blood cells;
step three, collecting umbilical cord blood plasma, centrifuging the transfer bag from which the red blood cells are removed in the step three again, transferring the supernatant into a new bag, namely collecting the plasma; freezing and storing the hematopoietic stem cells according to the umbilical cord blood freezing and storing process rule;
step four, preparing platelet-rich plasma, centrifuging the plasma obtained in the step above to remove red blood cells, collecting supernate, centrifuging again, and taking precipitate to obtain the platelet-rich plasma of the umbilical cord blood;
and step five, identifying the platelet-rich plasma and hematopoietic stem cells, and carrying out flow detection and counting on the surface markers of the platelet-rich plasma obtained in the step four.
Further, in the embodiment of the present invention, in the fifth step, the platelet rich plasma and the hematopoietic stem cells obtained by separation are subjected to cell viability, fully automatic blood cell counter analysis and flow cytometry analysis for identifying the platelet rich plasma and the hematopoietic stem cells.
Example two
Referring to fig. 1, a method for extracting platelet-rich plasma based on preparation of cord blood hematopoietic stem cells comprises the following steps:
firstly, pretreating a specimen, and disinfecting the surface of the specimen to remove surface stains and possible bacteria from the specimen to enter blood;
removing red blood cells, adding hydroxyethyl starch into the umbilical cord blood specimen, wherein the hydroxyethyl starch (HES) can be combined with negative charges on erythrocyte membranes to enable concave surfaces of the red blood cells to be attached to increase sedimentation speed, and removing the red blood cells by utilizing the difference of sedimentation speeds of the red blood cells and white blood cells;
step three, collecting umbilical cord blood plasma, centrifuging the transfer bag from which the red blood cells are removed in the step three again, transferring the supernatant into a new bag, namely collecting the plasma; freezing and storing the hematopoietic stem cells according to the umbilical cord blood freezing and storing process rule;
step four, preparing platelet-rich plasma, centrifuging the plasma obtained in the step above to remove red blood cells, collecting supernate, centrifuging again, and taking precipitate to obtain the platelet-rich plasma of the umbilical cord blood;
and step five, identifying the platelet-rich plasma and hematopoietic stem cells, and carrying out flow detection and counting on the surface markers of the platelet-rich plasma obtained in the step four.
Further, in the embodiment of the present invention, in the second step, the cord blood sample is obtained by measuring the ratio of the cord blood volume: hydroxyethyl starch = 5: 1 the desired amount of hydroxyethyl starch is added. Centrifuging at 500rpm for 7min, and separating into three layers, upper layer plasma, middle layer stem cells, and lower layer red blood cells. The hemostatic clamp of the 150ml transfer bag was clamped and the stem cells and plasma were slowly squeezed into another 250ml transfer bag.
Further, in the third step of the present invention, the 250ml transfer bag of the upper step is centrifuged at 1800rpm for 5min and divided into three distinct layers, the upper plasma layer, the middle stem cells layer and a few red blood cells layer. The hemostatic clamp of the 150ml transfer bag was opened and the plasma in the upper layer of the 250ml transfer bag was slowly expressed and drained into the 150ml transfer bag.
Further, in the present example, in the fourth step, the collected plasma was centrifuged for 1500prm 5min, and this step again removed red blood cells; the collected plasma was centrifuged again at 2500prm for 10 min. Discarding yellow clear supernatant with the upper layer of the precipitate being more than 3cm, and the lower layer being platelet-rich plasma.
EXAMPLE III
Referring to fig. 1, a method for extracting platelet-rich plasma based on preparation of cord blood hematopoietic stem cells comprises the following steps:
firstly, pretreating a specimen, and disinfecting the surface of the specimen to remove surface stains and possible bacteria from the specimen to enter blood;
removing red blood cells, adding hydroxyethyl starch into the umbilical cord blood specimen, wherein the hydroxyethyl starch (HES) can be combined with negative charges on erythrocyte membranes to enable concave surfaces of the red blood cells to be attached to increase sedimentation speed, and removing the red blood cells by utilizing the difference of sedimentation speeds of the red blood cells and white blood cells;
step three, collecting umbilical cord blood plasma, centrifuging the transfer bag from which the red blood cells are removed in the step three again, transferring the supernatant into a new bag, namely collecting the plasma; freezing and storing the hematopoietic stem cells according to the umbilical cord blood freezing and storing process rule;
step four, preparing platelet-rich plasma, centrifuging the plasma obtained in the step above to remove red blood cells, collecting supernate, centrifuging again, and taking precipitate to obtain the platelet-rich plasma of the umbilical cord blood;
and step five, identifying the platelet-rich plasma and hematopoietic stem cells, and carrying out flow detection and counting on the surface markers of the platelet-rich plasma obtained in the step four.
Further, in the embodiment of the present invention, in the second step, the cord blood sample is obtained by measuring the ratio of the cord blood volume: hydroxyethyl starch = 5: 1 the desired amount of hydroxyethyl starch is added. Centrifuging at 500rpm for 7min, and separating into three layers, upper layer plasma, middle layer stem cells, and lower layer red blood cells. The hemostatic clamp of the 150ml transfer bag was clamped and the stem cells and plasma were slowly squeezed into another 250ml transfer bag.
Further, in the third step of the present invention, the 250ml transfer bag of the upper step is centrifuged at 1800rpm for 5min and divided into three distinct layers, the upper plasma layer, the middle stem cells layer and a few red blood cells layer. The hemostatic clamp of the 150ml transfer bag was opened and the plasma in the upper layer of the 250ml transfer bag was slowly expressed and drained into the 150ml transfer bag.
Further, in the present example, in the fourth step, the collected plasma was centrifuged for 1500prm 5min, and this step again removed red blood cells; the collected plasma was centrifuged again at 2500prm for 10 min. Discarding the upper layer of yellow clear supernatant over 2cm, and the lower layer is platelet-rich plasma.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. A method for extracting platelet-rich plasma based on preparation of umbilical cord blood hematopoietic stem cells is characterized by comprising the following steps: the method comprises the following steps:
firstly, pretreating a specimen, and disinfecting the surface of the specimen to remove surface stains and possible bacteria from the specimen to enter blood;
removing red blood cells, namely adding hydroxyethyl starch into the umbilical cord blood specimen, wherein the hydroxyethyl starch can be combined with negative charges on erythrocyte membranes to ensure that the concave surfaces of the red blood cells are attached to accelerate the sedimentation speed, and removing the red blood cells by utilizing the difference of the sedimentation speeds of the red blood cells and the white blood cells;
step three, collecting umbilical cord blood plasma, centrifuging the transfer bag from which the red blood cells are removed in the step three again, transferring the supernatant into a new bag, namely collecting the plasma; freezing and storing the hematopoietic stem cells according to the umbilical cord blood freezing and storing process rule;
step four, preparing platelet-rich plasma, centrifuging the plasma obtained in the step above to remove red blood cells, collecting supernate, centrifuging again, and taking precipitate to obtain the platelet-rich plasma of the umbilical cord blood;
and step five, identifying the platelet-rich plasma and hematopoietic stem cells, and carrying out flow detection and counting on the surface markers of the platelet-rich plasma obtained in the step four.
2. The method for extracting platelet-rich plasma based on the preparation of cord blood hematopoietic stem cells according to claim 1, wherein: in the second step, the cord blood specimen is prepared according to the volume of cord blood: hydroxyethyl starch = 5: 1 the desired amount of hydroxyethyl starch is added. Centrifuging at 500rpm for 7min, and separating into three layers, upper layer plasma, middle layer stem cells, and lower layer red blood cells. The hemostatic clamp of the 150ml transfer bag was clamped and the stem cells and plasma were slowly squeezed into another 250ml transfer bag.
3. The method for extracting platelet-rich plasma based on the preparation of cord blood hematopoietic stem cells according to claim 1, wherein: in the third step, the 250ml transfer bag in the upper step is centrifuged at 1800rpm for 5min and divided into three clear layers, namely upper plasma layer, middle stem cells and a little red blood cells in the lower layer. The hemostatic clamp of the 150ml transfer bag was opened and the plasma in the upper layer of the 250ml transfer bag was slowly expressed and drained into the 150ml transfer bag.
4. The method for extracting platelet-rich plasma based on the preparation of cord blood hematopoietic stem cells according to claim 1, wherein: in step four, the collected plasma was centrifuged for 1500prm 5min, this step again removing red blood cells; the collected plasma was centrifuged again at 2500prm for 10 min. Discarding the upper layer of yellow clear supernatant with a length of 2-3cm, and the lower layer is platelet-rich plasma.
5. The method for extracting platelet-rich plasma based on the preparation of cord blood hematopoietic stem cells according to claim 1, wherein: and in the fifth step, the separated platelet-rich plasma and hematopoietic stem cells of the umbilical cord blood are subjected to cell viability, full-automatic blood cell counter analysis and flow cytometry analysis, and are used for identifying the platelet-rich plasma and the hematopoietic stem cells of the umbilical cord blood.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115011556A (en) * | 2022-08-10 | 2022-09-06 | 山东省齐鲁干细胞工程有限公司 | Separation method of umbilical cord blood hematopoietic stem cells |
| CN115261314A (en) * | 2022-06-28 | 2022-11-01 | 吉林省拓华生物科技有限公司 | Method for preparing mononuclear cells and platelets |
| CN115386549A (en) * | 2022-08-03 | 2022-11-25 | 河南省遗传资源细胞库有限公司 | Cryopreservation method for umbilical cord red blood cells |
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| CN115386549A (en) * | 2022-08-03 | 2022-11-25 | 河南省遗传资源细胞库有限公司 | Cryopreservation method for umbilical cord red blood cells |
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