1. A preparation and culture method of placental stem cells is characterized by comprising the following steps:

1) screening a lying-in woman;

2) collecting a sample;

3) transporting the sample;

4) sample handover and coding;

5) detecting a sample;

6) sample pretreatment;

7) separating and preparing cells;

8) primary culture;

9) subculturing;

10) detecting cells;

11) establishing a cell information file for retrieval;

12) and uploading the cell information file.

2. The method for preparing and culturing placental stem cells according to claim 1, wherein 5ml of maternal peripheral blood is taken for detection of pathogenic microorganisms after maternal screening in step 1).

3. The method for preparing and culturing placental stem cells according to claim 1, wherein the step 2) of collecting the sample comprises the following steps:

1) after the fetus is delivered out of the umbilical cord, collecting 5ml of umbilical cord blood for detection of pathogenic microorganisms and the like;

2) after the placenta is delivered, washing away blood stain on the surface of the placenta by using normal saline;

3) the placenta was placed in a collection box containing 400-600ml of storage fluid.

4. The method for preparing and culturing placental stem cells according to claim 1, wherein the sample pretreatment in step 6) comprises the following steps:

1) after the sample is detected to be qualified, taking out the tire disc from the collecting box;

2) fully washing the placenta with physiological saline to clean residual blood;

3) disinfecting and soaking with 75% medical alcohol;

4) then rinsing with normal saline for 2-3 times.

5. The method for preparing and culturing placental stem cells according to claim 1, wherein the cells prepared in step 7) are isolated and comprise the following steps:

1) transferring the pretreated placenta into a workbench, making the smooth surface of a placenta fetus face upwards, and drawing a cross-shaped incision by taking the root of an umbilical cord as a center to gently peel off the amnion;

2) carefully separating the placenta lobules by using an ophthalmologic curved scissors, cleaning the separated placenta lobules by using normal saline again, and draining;

3) picking and shearing tissue blocks by using an ophthalmological curved scissors until the tissue blocks are minced, transferring the sheared tissue blocks into a 50ml centrifugal tube, adding 0.05-0.25% enzyme containing 5-20 times of volume for digestion for 10-120min, adding 5-10 times of volume of normal saline to stop digestion, blowing and uniformly mixing according to the weight ratio of 1: (1-3), the mixture is slowly added into the lymphocyte separation liquid which is well packaged, the centrifuge is lifted and lowered by 1, the temperature is 2000rpm at 1000-.

6. The method for preparing and culturing placental stem cells according to claim 1, wherein the primary culture in step 8) comprises the following steps:

1) resuspending the treated tissue block or cell by using a mesenchymal stem cell primary culture solution, and then inoculating the tissue block or cell into a culture dish, wherein the mesenchymal stem cell primary culture solution should submerge the cell or tissue block by 2-5 mm;

2) placing the culture dish inoculated with the tissue block or the cell at 37 ℃ and 5% CO₂Culturing in an incubator, observing every 3 days, and supplementing or replacing liquid every 5-7 days according to cell climbing-out conditions.

7. The method for preparing and culturing placental stem cells according to claim 1, wherein the subculturing in step 9) comprises the following steps:

1) removing the culture solution, adding proper amount of normal saline, cleaning for 1-3 times, and thoroughly removing the residual tissue blocks;

2) adding 1-5ml stem cell mild enzyme or pancreatin, digesting at room temperature for 1-5min, and adding 5 times volume of physiological saline to stop digestion;

3) collecting cell suspension, centrifuging at 1200-1700rpm for 3-15min, discarding supernatant, and marking the obtained cell precipitate as P0 generation cells;

4) inoculating the P0 generation cells into a 150mm culture dish containing 20-25ml of serum-free complete culture medium, and adding CO₂The concentration is 5 percent, and the culture is continued in an incubator at the temperature of 37 ℃;

5) after the cell proliferation fusion degree reaches 85-95%, digesting and collecting to obtain P1 generation cells according to the processing steps;

6) inoculating the collected P1 generation cells into a 150mm culture dish containing 20-25ml of serum-free complete medium, and placing CO₂Continuing culturing in an incubator at 37 ℃ and the concentration of 5 percent until the cell proliferation fusion degree reaches 85-95 percent, and digesting according to the treatment steps to obtain P2 generation cells;

7) repeating the steps to obtain P3 and P4-PN generation cells.

8. The method for preparing and culturing placental stem cells according to claim 1, wherein the step 10) of detecting the cells comprises the steps of:

1) detecting bacteria, endotoxin and mycoplasma in the cell culture supernatant in the culture process;

2) after cell harvest, harvested cells were counted using a hemocytometer and cell activity was determined using trypan blue staining, while cell surface markers were detected using a flow cytometer.

9. The method for preparing and culturing placental stem cells according to claim 1, wherein said steps 11) and 12) of creating a retrievable cell information file and uploading the cell information file comprise the steps of:

1) numbering according to a group of systems of each person;

2) and filing paper versions of related information including sample sources, sample information, codes, preparation and culture information and the like, and uniformly inputting the paper versions into a computer for recording.

Background

Stem cell therapy technology is an emerging technology in the field of life sciences in recent years, has been applied to the therapeutic research of various intractable diseases, and has made active progress. Currently, the global stem cell therapy market can be divided into thalassemia, cerebral palsy, diabetes, leukemia, autism, and the like. With the annual increase in the incidence of many diseases such as diabetes and the like, the clinical demand for stem cells is increased, thereby promoting the development of the stem cell therapy market.

The placenta is an organ for exchanging substances between a fetus and a mother body, originates from an extraembryonic mesoderm in an embryonic development stage, is composed of mesenchyme, blood vessels and trophoblasts, and contains a large amount of mesenchyme components. The stem cell extracted from placenta has cell morphology, proliferation capacity, surface antigen mark, gene expression, etc. similar to that of marrow mesenchymal stem cell and has also multipotency of differentiation.

The placenta source stem cells are taken from placenta tissues after production, the collection process does not cause any damage to the mother and the neonate, the source is not limited by ethics and clinical medicine, and the placenta source stem cells are ideal stem cell resources.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation and culture method of the placental stem cells, which can improve the safety of the stem cells and simplify the operation steps.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a preparation and culture method of placental stem cells comprises the following steps:

1) screening a lying-in woman;

2) collecting a sample;

3) transporting the sample;

4) sample handover;

5) detecting a sample;

6) sample pretreatment;

7) separating and preparing cells;

8) primary culture;

9) subculturing;

10) detecting cells;

11) establishing a cell information file for retrieval;

12) and coding and uploading the cell information file.

Further, the screening of the puerpera in the step 1) refers to taking 5ml of puerpera peripheral blood to perform pathogenic microorganism detection after fully knowing that the condition of the family medical history and the sojourn history of the puerpera and the husband thereof meets the condition of a donor, wherein the pathogenic microorganism detection includes but is not limited to infectious disease detection such as hepatitis B, hepatitis C, syphilis, AIDS and the like.

Further, the step 2) of collecting the sample is to aseptically collect the placenta of a healthy newborn with normal term delivery, and comprises the following steps:

1) after the fetus is delivered out of the umbilical cord, collecting 5ml of umbilical cord blood for detection of pathogenic microorganisms and the like;

2) after the placenta is delivered, washing away blood stain on the surface of the placenta by using normal saline;

3) placing the placenta in a collection box filled with 400-600ml of storage liquid;

further, the storage liquid is normal saline containing gentamicin of 40 UI/ml.

Further, the sample transportation in the step 3) is to transfer the collected placenta collection box to a vaccine box at 4-8 ℃ for storage, and transport the placenta collection box to a storage place within 12-48 hours.

Further, the sample handover in the step 4) is to inspect the sample by a sample receiving person after the sample is transported to a storage place, observe whether the sample is abnormal, register related information and encode the information.

Further, the sample detection in step 5) is to perform sterility detection on the placenta, including but not limited to mycoplasma, fungi, bacteria, microorganisms, and the like, and specifically, the sample detection is performed by opening the collection box to draw the stock solution and/or taking the supernatant of the last centrifugation and washing in step 7).

Further, the sample pretreatment in the step 6) comprises the following steps:

1) taking out the tire disc from the collection box after the sample is qualified;

2) fully washing the placenta with physiological saline to clean residual blood;

3) disinfecting and soaking with 75% medical alcohol;

4) then rinsing with normal saline for 2-3 times.

Further, the cell separation preparation in the step 7) comprises the following steps:

1) transferring the pretreated placenta into a workbench, enabling the fetal surface (smooth surface) of the placenta to be upward, and drawing a cross-shaped incision by taking the root of an umbilical cord as the center to gently peel off the amnion;

2) carefully separating the placenta lobules by using an ophthalmologic curved scissors, cleaning the separated placenta lobules by using normal saline again, and draining;

3) picking and shearing tissue blocks by using an ophthalmological curved scissors until the tissue blocks are minced, transferring the sheared tissue blocks into a 50ml centrifugal tube, adding 0.05-0.25% enzyme containing 5-20 times of volume for digestion for 30-120min, adding 5-10 times of volume of normal saline to stop digestion, blowing and uniformly mixing according to the weight ratio of 1: (1-3), the mixture is slowly added into the separated lymphocyte solution, the centrifuge is lifted to 1, the temperature is 2000rpm, the temperature is 4 ℃ for 20 minutes, the cells on the middle white membrane layer and the white membrane layer are taken, the supernatant is removed by two times of centrifugal washing by physiological saline, the centrifuge is lifted to 9, the temperature is 2500rpm, the temperature is 4 ℃ for 3-10 minutes.

Further, the enzyme is preferably selected to be 0.1% collagenase IV.

Furthermore, when the tissue block is treated by the ophthalmologic curved scissors, the curved scissors are adopted for picking up the tissue block for 10-40min and the tissue block is cut to be 0.5-1.2mm³Preferably, the size of the tissue fragments is small.

Further, the primary culture in the step 8) comprises the following steps:

1) resuspending the treated tissue block or cell with a proper amount of mesenchymal stem cell primary culture solution, and inoculating the tissue block or cell into a culture dish, wherein the mesenchymal stem cell primary culture solution should submerge the cell or tissue block by 2-5 mm;

2) placing the culture dish inoculated with the tissue block or the cell at 37 ℃ and 5% CO₂Culturing in an incubator, observing every 3 days, and supplementing or replacing liquid every 5-7 days according to cell climbing-out conditions.

Furthermore, the primary culture solution of the mesenchymal stem cells is preferably a serum-free culture solution added with stem cell culture factors, and in order to further prevent cell contamination caused by improper operation, gentamicin (40UI/ml) needs to be added into the primary culture solution of the mesenchymal stem cells.

Further, the cellular digestion preferentially selects stem cell mild enzymes; the amount of cell digestive enzyme is preferably such that it covers all cells; the cell digestion time is preferably that the cells become round and slide down in a sand shape.

Further, the subculture in the step 9) is carried out after the cell crawl-out fusion degree reaches 25-40%, and the method comprises the following steps:

1) removing the culture solution, adding proper amount of normal saline, cleaning for 1-3 times, and thoroughly removing the residual tissue blocks;

2) adding 1-5ml stem cell mild enzyme or pancreatin, digesting at room temperature for 1-5min, and adding 5 times volume of physiological saline to stop digestion;

3) collecting cell suspension, centrifuging at 1200-1700rpm for 3-15min, discarding supernatant, and marking the obtained cell precipitate as P0 generation cells;

4) inoculating the P0 generation cells into a 150mm culture dish containing 20-25ml of serum-free complete culture medium, and adding CO₂The concentration is 5 percent, and the culture is continued in an incubator at the temperature of 37 ℃;

5) after the cell proliferation fusion degree reaches 85-95%, digesting and collecting to obtain P1 generation cells according to the processing steps;

6) inoculating the collected P1 generation cells into a 150mm culture dish containing 20-25ml of serum-free complete medium, and placing CO₂Continuing culturing in an incubator at 37 ℃ and the concentration of 5 percent until the cell proliferation fusion degree reaches 85-95 percent, and digesting according to the treatment steps to obtain P2 generation cells;

7) repeating the steps to obtain P3 and P4-PN generation cells.

Further, in order to further prevent cell contamination caused by mishandling, the serum-free complete medium needs to be supplemented with gentamicin (40 UI/ml).

Further, the cell detection in the step 10) comprises the following steps:

1) detecting bacteria, endotoxin and mycoplasma in the cell culture supernatant in the culture process;

2) after cell harvest, harvested cells were counted using a hemocytometer and cell activity was determined using trypan blue staining, while cell surface markers were detected using a flow cytometer.

Further, the step 11) and the step 12) of establishing a cell information file for retrieval, encoding and uploading the cell information file comprise the following steps:

1) numbering according to a group of systems of each person;

2) and filing paper versions of related information including sample sources, sample information, codes, preparation and culture information and the like, and uniformly inputting the paper versions into a computer for recording.

The invention adopts the structure to obtain the following beneficial effects: the preparation and culture method of the placental stem cells has the advantages of strong cell proliferation and differentiation capacity, less impurity cells, relative purity, simple separation and culture process, small workload and high cell climbing probability, can harvest a large amount of mesenchymal stem cells, and meets the requirements of mass production of stem cell products and clinical application.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 1

A preparation and culture method of placental stem cells comprises the following steps:

1) screening a lying-in woman;

2) collecting a sample;

3) transporting the sample;

4) sample handover and coding;

5) detecting a sample;

6) sample pretreatment;

7) separating and preparing cells;

8) primary culture;

9) subculturing;

10) detecting cells;

11) establishing a cell information file for retrieval;

12) and upload the cell information file.

The screening of the lying-in woman in the step 1) refers to taking 5ml of lying-in woman peripheral blood to carry out pathogenic microorganism detection including but not limited to infectious disease detection such as hepatitis B, hepatitis C, syphilis, AIDS and the like after fully knowing that the condition of the family medical history and the sojourn history of the lying-in woman and the husband thereof meets the condition of a donor.

The step 2) of collecting the sample is to aseptically collect the placenta of a healthy newborn delivered at normal term, and comprises the following steps:

1) after the fetus is delivered out of the umbilical cord, collecting 5ml of umbilical cord blood for detection of pathogenic microorganisms and the like;

2) after the placenta is delivered, washing away blood stain on the surface of the placenta by using normal saline;

3) placing the placenta in a collection box filled with 400-600ml of storage liquid;

and in the step 3), the sample transportation is to transfer the collected placenta collection box to a vaccine box at 4-8 ℃ for storage, and transport the placenta collection box to a storage place within 12-48 hours.

And in the step 4), sample handover refers to that after the sample is transported to a storage place, a sample receiving person checks the sample, observes whether the sample is abnormal or not, registers related information and codes.

The sample detection in the step 5) is to perform sterility detection on the placenta, including but not limited to mycoplasma, fungi, bacteria, microorganisms and the like, and specifically, the detection is performed by opening the collection box and extracting the storage solution.

The sample pretreatment in the step 6) comprises the following steps:

1) taking out the tire disc from the collection box after the sample is qualified;

2) fully washing the placenta with physiological saline to clean residual blood;

3) disinfecting and soaking with 75% medical alcohol;

4) then rinsing with normal saline for 2-3 times.

The cell separation preparation in the step 7) comprises the following steps:

1) transferring the pretreated placenta to a workbench, making the fetal surface of the placenta and the smooth surface upward, and making a cross incision by taking the root of an umbilical cord as the center to gently peel off the amnion;

2) carefully separating the placenta lobules by using an ophthalmologic curved scissors, cleaning the separated placenta lobules by using normal saline again, and draining;

3) picking and shearing tissue blocks by using an ophthalmological curved scissors until the tissue blocks are minced, transferring the sheared tissue blocks into a 50ml centrifugal tube, adding 0.05-0.25% enzyme containing 5-20 times of volume for digestion for 10-120min, adding 5-10 times of volume of normal saline to stop digestion, blowing and uniformly mixing according to the weight ratio of 1: (1-3), the mixture is slowly added into the lymphocyte separation liquid which is well packaged, the centrifuge is lifted and lowered by 1, the temperature is 2000rpm at 1000-.

The primary culture in the step 8) comprises the following steps:

1) resuspending the treated tissue block or cell with a proper amount of mesenchymal stem cell primary culture solution, and inoculating the tissue block or cell into a culture dish, wherein the mesenchymal stem cell primary culture solution should submerge the cell or tissue block by 2-5 mm;

2) placing the culture dish inoculated with the tissue block or the cell at 37 ℃ and 5% CO₂Culturing in an incubator, observing every 3 days, and supplementing or replacing liquid every 5-7 days according to cell climbing-out conditions.

The subculture in the step 9) is to perform subculture after the cell crawl-out fusion degree reaches 25-40%, and the method comprises the following steps:

1) removing the culture solution, adding proper amount of normal saline, cleaning for 1-3 times, and thoroughly removing the residual tissue blocks;

2) adding 1-5ml stem cell mild enzyme or pancreatin, digesting at room temperature for 1-5min, and adding 5 times volume of physiological saline to stop digestion;

3) collecting cell suspension, centrifuging at 1200-1700rpm for 3-15min, discarding supernatant, and marking the obtained cell precipitate as P0 generation cells;

4) inoculating the P0 generation cells into a 150mm culture dish containing 20-25ml of serum-free complete culture medium, and adding CO₂The concentration is 5 percent, and the culture is continued in an incubator at the temperature of 37 ℃;

5) after the cell proliferation fusion degree reaches 85-95%, digesting and collecting to obtain P1 generation cells according to the processing steps;

6) inoculating the collected P1 generation cells into a 150mm culture dish containing 20-25ml of serum-free complete medium, and placing CO₂Continuing culturing in an incubator at 37 ℃ and the concentration of 5 percent until the cell proliferation fusion degree reaches 85-95 percent, and digesting according to the treatment steps to obtain P2 generation cells;

7) repeating the steps to obtain P3 and P4-PN generation cells.

The cell detection in the step 10) comprises the following steps:

1) detecting bacteria, endotoxin and mycoplasma in the cell culture supernatant in the culture process;

2) after cell harvest, harvested cells were counted using a hemocytometer and cell activity was determined using trypan blue staining, while cell surface markers were detected using a flow cytometer.

The step 11) and the step 12) of establishing a cell information file for retrieval, coding and uploading the cell information file comprise the following steps:

1) numbering according to a group of systems of each person;

2) and filing paper versions of related information including sample sources, sample information, codes, preparation and culture information and the like, and uniformly inputting the paper versions into a computer for recording.

Example 2

A preparation and culture method of placental stem cells comprises the following steps: the method comprises the following steps of screening a puerpera, collecting a sample, transporting the sample, handing over the sample, detecting the sample, preprocessing the sample, separating and preparing cells, primary culture, subculture, detecting the cells, establishing a cell information archive for retrieval, coding and uploading the cell information archive, and specifically comprises the following steps:

1) screening a puerpera: firstly, investigating the family medical history and sojourn history of a lying-in woman and a husband thereof, and taking 5ml of lying-in woman peripheral blood to carry out pathogenic microorganism detection including but not limited to infectious disease detection such as hepatitis B, hepatitis C, syphilis, AIDS and the like after meeting the donor conditions;

2) collecting samples: aseptically collecting placenta of normal term parturition healthy newborn, which comprises collecting 5ml umbilical blood after breaking umbilicus for pathogenic microorganism detection including but not limited to infectious disease detection of hepatitis B, hepatitis C, syphilis, AIDS, etc.; cutting off the umbilical cord at a position 3-5cm away from the placenta after the placenta is delivered, and placing the placenta with the umbilical cord removed in a collection box filled with 400-600ml of storage liquid; storing the collection box at 4 ℃, and conveying the collection box to a company for registering relevant information within 12 hours;

3) sample transportation: transferring the collected placenta collection box to a vaccine box at 4-8 ℃ for storage, and conveying to a storage place within 12-48 hours;

4) sample handover: after the sample is transported to a storage place, a sample receiving person checks the sample, observes whether the sample is abnormal or not, registers related information and codes;

5) sample detection: after receiving the sample, the sample receiver checks whether the sample is abnormal, hands the sample to a microbiological laboratory for infectious disease and sterile detection, and hands the sample to a laboratory preparation person for processing under the condition that the sample is abnormal;

6) sample pretreatment: after the sample is detected to be qualified, taking out the placenta from the collecting box, fully washing the placenta with normal saline to clean residual blood, disinfecting and soaking the placenta with 75% medical alcohol, and rinsing the placenta for 2-3 times with the normal saline;

7) cell separation preparation: the method comprises the following steps of enabling the surface (smooth surface) of a fresh placenta fetal to face upwards, scratching a cross cut with the root of an umbilical cord as the center, slightly stripping the amnion, after stripping the amnion, carefully separating placenta lobules, cleaning the stripped placenta lobules again with normal saline, draining, shearing into minced meat by using bending scissors in ophthalmology, transferring the minced meat into a 50ml centrifugal tube, adding 0.25% collagenase containing 5 times of volume for digestion for 30min, adding 10 times of volume of normal saline to stop digestion, blowing and uniformly mixing, and according to the ratio of 1: 1, the mixture is slowly added into the separated lymphocyte liquid, the centrifuge is lifted to 1 and lowered to 2000rpm, the mixture is centrifuged for 20 minutes at 4 ℃, cells on a middle leucocyte layer and a leucocyte layer are taken, and the cells are centrifuged and washed twice by physiological saline (the centrifuge is lifted to 9 and lowered to 9, the rpm is 2000, the mixture is centrifuged for 5 minutes at 4 ℃);

8) primary culture: adding 3 times of the volume of the mesenchymal stem cell primary culture solution into the resuspended tissue block or cell, inoculating the tissue block or cell into a culture dish, determining whether to supplement the culture solution according to the condition that the culture solution covers the tissue or cell, ensuring that the mesenchymal stem cell primary culture solution is 2mm higher than the cell or tissue block, shaking uniformly, transferring the culture dish to 37 ℃, and then 5% CO₂Culturing in an incubator, observing once every 3 days, and supplementing or replacing liquid every 5-7 days according to cell climbing-out conditions;

9) subculturing: and (3) carrying out passage after the cell crawl-out fusion degree reaches 25-40%, which comprises the following steps: carefully removing the culture solution, adding a proper amount of normal saline, washing for 2 times to thoroughly remove the residual tissue blocks, adding 3ml of stem cells, carrying out mild enzyme digestion for 3min, and adding 5 times volume of normal saline to stop digestion; collecting cell suspension, centrifuging at 1200rpm for 10min, discarding supernatant, and recording the obtained cell precipitate as P0 generation cells; the P0 generation cells were seeded into a 150mm dish containing 20ml serum-free complete medium and placed in CO₂Culturing at 5% concentration and 37 deg.C in incubator until cell proliferation fusion degree reaches 85-95%, and digesting and collecting to obtain P1 generation cells; inoculating the collected P1 generation cells into a 150mm culture dish containing 20-25ml of serum-free complete medium, and placing CO₂Culturing at 5% concentration and 37 deg.C in incubator until cell proliferation fusion degree reaches 85-95%, and digesting according to the above steps to obtain P2 generation cell; repeating the steps to obtain P3 and P4-PN generation cells;

10) cell detection: the cell detection is to detect bacteria, endotoxin, mycoplasma and the like in the cell culture supernatant in the cell culture process; after the cells are harvested, counting the harvested cells by using a blood counting plate, measuring the activity of the cells by using trypan blue staining, and detecting cell surface markers by using a flow cytometer;

11) the cell information archives which can be searched are established and uploaded according to a group of system numbers of each person, and relevant information including sample sources, sample information, codes, preparation and culture information and other paper versions are filed and uniformly input into a computer for recording and used for management and searching.

The present invention and its embodiments have been described above, but the description is not limitative, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.