CN106834230A - A kind of preparation method of placental hematopoietic stem cell - Google Patents
A kind of preparation method of placental hematopoietic stem cell Download PDFInfo
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- CN106834230A CN106834230A CN201710111925.7A CN201710111925A CN106834230A CN 106834230 A CN106834230 A CN 106834230A CN 201710111925 A CN201710111925 A CN 201710111925A CN 106834230 A CN106834230 A CN 106834230A
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- placenta
- stem cell
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- hematopoietic stem
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention provides a kind of preparation method of placental hematopoietic stem cell, the preparation method includes placenta pretreatment, placental blood collection, the purification of placental hematopoietic stem cell, the placental hematopoietic stem cell infection rate that the preparation method is obtained is low, and cell quantity is more, and extraction step is simple to operate.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of preparation method of placental hematopoietic stem cell.
Background technology
Candidate stem cell, refers to the class cell with self-renewing and Multidirectional Differentiation ability.Its fundamental characteristics is tool
Have self-renewal capacity, i.e., by a cell cycle events after, can produce two with division before property identical hematopoiesis
Stem cell, while having Multidirectional Differentiation ability again, i.e., under certain environmental conditions, candidate stem cell has to each system's haemocyte
The ability of differentiation, is widely used in malignant hematologic disease（Such as acute leukemia, chronic myelocytic leukemia）, it is non-malignant intractable
Blood disease（Such as alpastic anemia, myelodysplastic syndrome）Genetic disease（In congenital immune deficiency Bing ﹑ ground
Extra large anaemia etc.）With some treatment of solid tumors.HSCT refers to carry out full-body exposure, chemotherapy and immunosupress to patient
After pretreatment, Normal donor or autologous candidate stem cell intravascular are transfused to patient, make the normal hematopoiesis of reconstruction and be immunized
Function.
With the development of medical science and biotechnology, substantial amounts of candidate stem cell is contained in discovered in recent years placenta, and it is contained
Candidate stem cell quantity is very high, and the distribution type requirement of transplanting placental hematopoietic stem cell need not be very strict, reacted after transplanting compared with
It is light and need not using medicine the advantages of, the method for extracting candidate stem cell from placenta tissue turns into the research direction in field.
The content of the invention
A kind of present invention collects candidate stem cell method in providing mature placenta from healthy puerpera.
Methods described includes the pretreatment of placenta, placental blood collection, the purification of placental hematopoietic stem cell, and step is respectively：
1st, placenta pretreatment：In an aseptic environment, placenta is pressed from both sides out from collecting cassette with aseptic nipper and is put into preprepared
In aseptic can, and one end of placenta surface umbilical cord is lived with sterility forceps sub-folder, remove amnion, the clot of placenta surface attachment, used
Phosphate buffer PBS rinses placenta surface and soaks one minute, and amount of buffer should not have whole placenta, rinses placenta number of times and exists
More than 2 times, and often rinse once all should separately change one by sterilizing container, in phosphate buffer PBS add combination antibiosis
Element.
2nd, placental blood is collected：It is mixed with the anticoagulant for storage of whole blood of physiological saline and volume fraction 10% after having cleaned placenta surface
Close liquid disposable syringe and push placenta blood vessel from umbilical cord arteria umbilicalis inserting needle, collect the placenta blood of outflow, the blood that will be collected
Liquid is transferred in centrifuge tube.
Continuation pours into placenta with the physiological saline containing the AMD3100 that mass concentration is 10g/L, and clamping navel with umbilical cord clamps moves
Arteries and veins, after being incubated 30 minutes, collects flushing liquor.
3rd, the purification of placental hematopoietic stem cell：By for the first time and after the placental blood mixing of second collection, formed sediment by ethoxy
Powder and placental blood ratio are 4:1 adds HES to mix 15 minutes, dispenses to centrifuge tube be centrifuged respectively, centrifugal condition
For：Centrifugation rate is 50g/min, and 5 grades of raising speed, 1 grade of reduction of speed, centrifugation time 15min, centrifugation collects supernatant after terminating, will be upper
Clear liquid is centrifuged again, and centrifugal condition is：Rotating speed 2000rpm/min, accelerates 7 grades, 4 grades of reduction of speed, centrifugation time 10min, centrifugation knot
Centrifugation bottom of the tube karyocyte is collected after beam.Cell after being centrifuged with RPMI1640 mix suspendings, is carried out with common detection methods
The counting of candidate stem cell, Activity determination；In adding methylcellulose medium in cell suspension, and vibration to bubble disappears
Dissipate, be inoculated in Tissue Culture Plate, be put into 37 DEG C, cultivated in the CO2gas incubator of 5% concentration, routine observation cell colony
Formation state.
The component of the phosphate buffer PBS of placenta pretreatment is in above-mentioned steps 1：Added in 1000ml sterilizing ultra-pure waters
Sodium chloride 8.5g, disodium hydrogen phosphate 2.85g, potassium dihydrogen phosphate 0.27g, potassium chloride 0.2g, wherein add antibiotic in buffer solution
Combination penicillin, streptomysin mixed liquor（It is dual anti-）, to add concentration respectively be 3%, 2%, 1% for gentamicin, amphotericin B.
Beneficial effects of the present invention：
The placental hematopoietic stem cell infection rate obtained by the preparation method of placental hematopoietic stem cell of the present invention is low, cell
Quantity is more, and extraction step is simple to operate.
Brief description of the drawings
Fig. 1 is FCM analysis figure；
Fig. 2：Placenta blood cell colony form 40 ×, wherein A is the quick-fried formula colony of red blood cell that placental blood is formed, and B is placental blood shape
Into GMC.
Specific embodiment
For a further understanding of the present invention, this breaking-out is further illustrated with reference to embodiment, but not as to this hair
Bright restriction.
1st, placenta pretreatment：In an aseptic environment, placenta is pressed from both sides out from collecting cassette with aseptic nipper and is put into preparation in advance
In good aseptic can, and one end of placenta surface umbilical cord is lived with sterility forceps sub-folder, remove amnion, the blood of placenta surface attachment
Block, rinses placenta surface and soaks one minute with phosphate buffer PBS, and amount of buffer should not have whole placenta, rinse placenta
Number of times is often rinsed and once all should separately change a container by sterilizing more than 2 times, and the component of phosphate buffer PBS is：
Sodium chloride 8.5g, disodium hydrogen phosphate 2.85g, potassium dihydrogen phosphate 0.27g, potassium chloride 0.2g are added in 1000ml sterilizing ultra-pure waters,
Antibiotics combination combination A penicillin, streptomysin mixed liquor are added wherein in buffer solution（It is dual anti-）, addition concentration is 1%, final
Pollution rate to placental blood candidate stem cell is 5%；Addition antibiotic combinations B penicillin, streptomysin mixed liquor（It is dual anti-）, celebrating it is big
Mycin adds concentration and is respectively 2% and 1% respectively, and the pollution rate for finally giving placental blood candidate stem cell is 3%；Addition antibiotic
Combination C penicillin, streptomysin mixed liquor（It is dual anti-）, to add concentration respectively be 3%, 2%, 1%, every group for gentamicin, amphotericin B
50 placentas of experiment, isolated placental hematopoietic stem cell, separately sampled use hemoculture instrument instrument culture finally gives placenta
The pollution rate of blood candidate stem cell is less than 1%.
2nd, placental blood is collected：After having cleaned placenta surface, with 100mL physiological saline and the blood of volume fraction 10% purchased in market
The mixed liquor disposable syringe that liquid preserves liquid III pushes placenta blood vessel from umbilical cord arteria umbilicalis inserting needle, collects the placental blood of outflow
Liquid, the blood of collection is transferred in 50mL centrifuge tubes.
Continuation pours into placenta with 100 milliliters of the physiological saline containing the AMD3100 that mass concentration is 10g/L, is pressed from both sides with umbilical cord clamps
Tight arteria umbilicalis, after being incubated 30 minutes, collects flushing liquor.
3rd, the purification of placental hematopoietic stem cell：By for the first time and after the placental blood mixing of second collection, formed sediment by ethoxy
Powder and placental blood ratio are 4:1 adds HES to mix 15 minutes, dispenses respectively to 50mL centrifuge tubes, and centrifugal condition is：
Centrifugation rate is 50g/min, and 5 grades of raising speed, 1 grade of reduction of speed, centrifugation time 15min, centrifugation collects supernatant after terminating, by supernatant
It is centrifuged again, centrifugal condition is：Rotating speed 2000rpm/min, accelerates 7 grades, 4 grades of reduction of speed, centrifugation time 10min, after centrifugation terminates
Collect centrifugation bottom of the tube karyocyte.
4th, the detection of placental hematopoietic stem cell：Cell after being centrifuged with RPMI1640 mix suspendings, uses common detection methods
Counting, the Activity determination of candidate stem cell, placental hematopoietic stem cell number of nucleated cells and CD34+ cell numbers such as table one are carried out, is flowed
Formula cell instrument detects CD34+ cell quantities as shown in figure 1, the ratio of CD34+ positive cells can reach 1%-2%.
Table one：
| Sample number | Sample volume（ml） | Number of nucleated cells | CD34+ cell numbers |
| 1 | 150 | ||
| 2 | 145 | ||
| 3 | 122 |
5th, cell colony culture：Take 0.9 milliliter 5 × 104Cell suspension, add methylcellulose medium in, and vibrate extremely
Dissipation of air bubbles, is inoculated in Tissue Culture Plate, is put into 37 DEG C, is cultivated in the CO2gas incubator of 5% concentration, routine observation, carefully
Born of the same parents are inoculated with and cell colony can be observed within the 14th day and formed well, as shown in Figure 2.Placental hematopoietic stem cell cell colony forms number
Such as table two.
Table two：
| Sample number | The quick-fried formula colony of red blood cell | GMC |
| 1 | 55 | 65 |
| 2 | 42 | 64 |
| 3 | 32 | 60 |
Claims (2)
1. a kind of preparation method of placental hematopoietic stem cell, it is characterised in that the preparation method includes placenta pretreatment, placenta
Blood is collected, the purification of placental hematopoietic stem cell, and step is：
1）, placenta pretreatment：In an aseptic environment, placenta is pressed from both sides out from collecting cassette with aseptic nipper and is put into preprepared
In aseptic can, and one end of placenta surface umbilical cord is lived with sterility forceps sub-folder, remove amnion, the clot of placenta surface attachment, used
Phosphate buffer PBS rinses placenta surface and soaks one minute, and amount of buffer should not have whole placenta, rinses placenta number of times and exists
More than 2 times, and often rinse once all should separately change one by sterilizing container, in phosphate buffer PBS add combination antibiosis
Element；
2）, placental blood collect：After having cleaned placenta surface, with the mixing of physiological saline and the anticoagulant for storage of whole blood of volume fraction 10%
Liquid disposable syringe pushes placenta blood vessel from umbilical cord arteria umbilicalis inserting needle, collects the placenta blood of outflow, the blood that will be collected
It is transferred in centrifuge tube；
Continuation pours into placenta with the physiological saline containing the AMD3100 that mass concentration is 10g/L, and arteria umbilicalis is clamped with umbilical cord clamps, incubates
After educating 30 minutes, flushing liquor is collected；
3）, placental hematopoietic stem cell purification：Will for the first time and after second placental blood collected mixes, by HES with
Placental blood ratio is 4:1 adds HES to mix 15 minutes, dispenses to centrifuge tube be centrifuged respectively, and centrifugal condition is：
Centrifugation rate is 50g/min, and 5 grades of raising speed, 1 grade of reduction of speed, centrifugation time 15min, centrifugation collects supernatant after terminating, by supernatant
It is centrifuged again, centrifugal condition is：Rotating speed 2000rpm/min, accelerates 7 grades, 4 grades of reduction of speed, centrifugation time 10min, after centrifugation terminates
Collect centrifugation bottom of the tube karyocyte；Cell after being centrifuged with RPMI1640 mix suspendings, hematopoiesis is carried out with common detection methods
The counting of stem cell, Activity determination；In adding methylcellulose medium in cell suspension, and vibrate to dissipation of air bubbles, connect
Plant in Tissue Culture Plate, be put into 37 DEG C, cultivated in the CO2gas incubator of 5% concentration, routine observation cell colony forms shape
State.
2. the preparation method of placental hematopoietic stem cell as claimed in claim 1, it is characterised in that the phosphorus of the placenta pretreatment
The component of phthalate buffer PBS is：Sodium chloride 8.5g, disodium hydrogen phosphate 2.85g, di(2-ethylhexyl)phosphate are added in 1000ml sterilizing ultra-pure waters
Hydrogen potassium 0.27g, potassium chloride 0.2g, add antibiotic combinations penicillin, streptomysin mixed liquor wherein in buffer solution（It is dual anti-）, celebrating it is big
It is 3%, 2%, 1% that mycin, amphotericin B add concentration respectively.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710111925.7A CN106834230A (en) | 2017-02-28 | 2017-02-28 | A kind of preparation method of placental hematopoietic stem cell |
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| CN201710111925.7A CN106834230A (en) | 2017-02-28 | 2017-02-28 | A kind of preparation method of placental hematopoietic stem cell |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110387349A (en) * | 2018-04-23 | 2019-10-29 | 医晟生医股份有限公司 | Promote the method and its product of bioactie agent in placenta tissue |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103756965A (en) * | 2014-01-27 | 2014-04-30 | 山东省齐鲁干细胞工程有限公司 | Method for lavaging hematopoietic stem cells from placenta |
| CN103789262A (en) * | 2014-02-17 | 2014-05-14 | 宁波普莱森特生物科技有限公司 | Preparation method and preservation method of clinical application-level placental hematopoietic stem cells |
| CN104711226A (en) * | 2015-04-09 | 2015-06-17 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method of placenta hematopoietic stem cells |
| CN105695302A (en) * | 2016-03-14 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Filling and acquiring devices for extracting hematopoietic stem cells from placenta |
-
2017
- 2017-02-28 CN CN201710111925.7A patent/CN106834230A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103756965A (en) * | 2014-01-27 | 2014-04-30 | 山东省齐鲁干细胞工程有限公司 | Method for lavaging hematopoietic stem cells from placenta |
| CN103789262A (en) * | 2014-02-17 | 2014-05-14 | 宁波普莱森特生物科技有限公司 | Preparation method and preservation method of clinical application-level placental hematopoietic stem cells |
| CN104711226A (en) * | 2015-04-09 | 2015-06-17 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method of placenta hematopoietic stem cells |
| CN105695302A (en) * | 2016-03-14 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Filling and acquiring devices for extracting hematopoietic stem cells from placenta |
Non-Patent Citations (2)
| Title |
|---|
| 章涛等: "人胎盘组织造血干/祖细胞的分离富集", 《中国实验血液学杂志》 * |
| 莫峥等: "胎盘来源细胞与母血、脐血细胞的嵌合", 《中国组织工程研究》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110387349A (en) * | 2018-04-23 | 2019-10-29 | 医晟生医股份有限公司 | Promote the method and its product of bioactie agent in placenta tissue |
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Application publication date: 20170613 |