CN107326062A - A kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations - Google Patents
A kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations Download PDFInfo
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- CN107326062A CN107326062A CN201710492354.6A CN201710492354A CN107326062A CN 107326062 A CN107326062 A CN 107326062A CN 201710492354 A CN201710492354 A CN 201710492354A CN 107326062 A CN107326062 A CN 107326062A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/56988—HIV or HTLV
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
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Abstract
The invention discloses a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations, comprise the following steps：S1, a certain amount of (about 15ml) the bleeding of the umbilicus sample of extraction, carry out six detections of infectious disease；S2, will pass through six detect after bleeding of the umbilicus sample carry out freeze-stored cell product (P2 generations) examine and determine；S3, provided (recovery) cell calibrating；S4, on request complete pack, freeze, transporting and the term of validity demarcation.Can standardize, standardize the invention enables the assessment overall process of umbilical cord mesenchymal stem cells quality, sequencing, product can control by quality management system.
Description
Technical field
The present invention relates to medical field, and in particular to a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations.
Background technology
The species that mescenchymal stem cell is used for clinical application research is mainly mesenchymal stem cells MSCs and umbilical cord mesenchyma
Stem cell, this two classes cell all there is the characteristics of mescenchymal stem cell is common low immunogenicity, particularly the latter to become apparent.Navel
Institutional framework is simple in band, and except main big blood vessel, remaining causes umbilical cord mesenchymal stem cells based on connective tissue
Substantially not with antigen contact, so immunogenicity is very low, this significantly increases the possibility of clinical practice.Between umbilical cord source
Mesenchymal stem cells are not only able to the ideal substitute as mesenchymal stem cells MSCs, and with bigger application potential.
Preparation of the more report on umbilical cord mesenchymal stem cells through having.The mesenchyma for how quickly and efficiently preparing high-quality is done
It is one of hot research content of current stem cell that cell, which is used for clinical and scientific research,.It is dry thin that umbilical cord mesenchyma is not yet related at present
The data of the method for evaluating quality of born of the same parents' preparation is disclosed.
The content of the invention
To solve the above problems, the invention provides a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations.
To achieve the above object, the technical scheme taken of the present invention is：
A kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations, comprises the following steps：
S1, a certain amount of (about 15ml) the bleeding of the umbilicus sample of extraction, carry out six detections of infectious disease；
S2, will pass through six detect after bleeding of the umbilicus sample carry out freeze-stored cell product (P2 generations) examine and determine；
S3, provided (recovery) cell calibrating；
S4, on request complete pack, freeze, transporting and the term of validity demarcation.
Wherein, it is described six detection include HbsAg, anti-HCV, syphilis helicoid antibody, anti-HIV, cytomegalovirus and
ALT detection；Wherein preceding four using ELISA detection, and testing result should be negative；Cytomegalovirus is exempted from using enzyme-linked
Epidemic disease method detects that IgG and IgM testing result should be negative；ALT detects that testing result answers≤40 units using performance rate method.
Wherein, the step S2 specifically includes following steps：
S21, Cell viability detection；
S22, the observation for carrying out by inverted microscope cellular morphology, it is thin that the attached cell of collection should be into fibrous fusiformis
Born of the same parents；
S23, mescenchymal stem cell phenotypic characteristic detection
Sample is arranged by bar code number order, if every part of sample according to detection antibody type the need for take streaming dedicated pipe
The Heavenly Stems and Earthly Branches, have marked Isotype control pipe and antibody pipe respectively；Sample is moved into special streaming pipe, 500g is centrifuged 5 minutes；Abandon supernatant,
1ml PBS are added, are fully mixed, 500g is centrifuged 5 minutes, abandons supernatant, the PBS suspension cells precipitation of proper volume is added；
The μ l cell suspensions of sample 100 are separately added into two pipes；Respective amount antibody is separately added into corresponding pipe and corresponding same
Type contrast agents；Fully mix, put 4 DEG C of refrigerator lucifuges and be incubated 30 minutes.1ml PBS are added, are fully mixed, 500g centrifuges 5 points
Clock；Supernatant is abandoned, 1mL PBSs is then added, fully mixes, 500g is centrifuged 5 minutes；Supernatant is abandoned, appropriate PBS is added,
Fully mix, adjustment cell to suitable concn, then upper machine testing；Directly detected in recovery freeze-stored cell or recovery is frozen if necessary
Deposit cell to be further cultured in detecting after 72h, the result of at least one test point should comply fully with claimed below：Positive antigen：
CD29, CD44, CD73, CD90, CD105 (wherein CD29, CD44 are used as sampling observation index, 1 times/year), positive indication expression rate >=
95%；Negative antigens：CD14, CD19, CD34, CD45, HLA-DR, negative indication expression rate≤2%；
S24, cell final wash supernatant 30ml is taken, according to《Sterility testing S.O.P.》Middle hemoculture instrument detection method
Sterility testing is carried out, testing result should meet regulation；
S25, the μ l of cell suspension 100 are taken, according to《Detection of mycoplasma S.O.P.》Carry out detection of mycoplasma, testing result
Regulation should be met；
S26, endotoxin detection：Cell suspension 0.5ml is taken, according to《Endotoxin examination criteria operation sequence》Detection, should meet
Regulation；
S27, cell suspension is taken, according to《Karyotyping S.O.P.》Karyotyping is carried out, cell should be in chromosome water
It is flat to keep stable；
S28, cell suspension is taken, according to《Genomic methylation examination criteria operation sequence》Genomic methylation detection is carried out,
The methylation level of genome should be unchanged；
S29, cell suspension is taken, according to《Telomerase activation examination criteria operation sequence》Carry out telomerase activation detection, cell
Telomerase activation should be unchanged.
Wherein, the step S3 specifically includes following steps：
Cell is collected in S31, digestion, and final concentration of 1~2 × 106/ml single cell suspension is made with sodium chloride injection,
Draw and blow and beat uniform after 10 μ l trypan blue solutions are mixed with 10 μ l samples, take 10 μ l mixing suspensions, with tally into oblique 45 degree of angles
Note in cover glass edge, repeat the action, another cell of tally is full of with remaining 10 μ l cell suspensions；It will count
Plate lies on microscopical objective table and counted；Single chamber, which counts cell, must not be less than 400；The blue dye cells on total cells number of observation
Ratio.Indigo plant dye cell number should be less than 15%, and Cell viability should be greater than 85%；
S32, cell final wash supernatant 30ml is taken, according to《Sterility testing S.O.P.》Middle hemoculture instrument detection method
Sterility testing is carried out, as a result should meet regulation；
S33, the cell provided every time, after packing terminates, take remnants cell suspension from (final step) separation container
A little, Gram's staining detection is carried out, should be without Gram-negative bacteria or gram-positive bacteria.
Wherein, the step S21 uses trypan blue staining, specifically includes following steps：Cell is collected in digestion, with chlorination
Sodium injection is made final concentration of 1~2 × 106/ ml single cell suspension, draws 10 μ l trypan blue solutions and is mixed with 10 μ l samples
Blow and beat uniformly afterwards, take 10 μ l mixing suspensions, with tally into oblique 45 degree of subscripts in cover glass edge, repeat the action, will count
Another cell of plate is full of with remaining 10 μ l cell suspensions；Tally is lain on microscopical objective table and counted；It is single
Room, which counts cell, must not be less than 400；The blue dye cells on total cells number ratio of observation；Indigo plant dye cell number should be less than 15%, cell
Motility rate should be greater than 85%.
Wherein, the step S21 is detected using CASY-TT rapid cellulars analyzer, and Cell viability should be greater than 85%.
The invention has the advantages that：
The assessment overall process of umbilical cord mesenchymal stem cells quality standardized, standardized, sequencing, product energy quilt
Quality management system is controlled.
Embodiment
In order that objects and advantages of the present invention are more clearly understood, the present invention is carried out with reference to embodiments further
Describe in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair
It is bright.
Embodiment
A kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations, comprises the following steps：
S1, a certain amount of (about 15ml) the bleeding of the umbilicus sample of extraction, carry out six detections of infectious disease；Six detections include
HbsAg, anti-HCV, syphilis helicoid antibody, anti-HIV, cytomegalovirus and ALT detection；Wherein preceding four are exempted from using enzyme-linked
Epidemic disease method detects that testing result should be negative；Cytomegalovirus detects that IgG and IgM testing result should be using ELISA
It is negative；ALT detects that testing result answers≤40 units using performance rate method.
S2, will pass through six detect after bleeding of the umbilicus sample carry out freeze-stored cell product (P2 generations) examine and determine；Specifically include as follows
Step：
S21, Cell viability detection；
Method one：Using trypan blue staining, following steps are specifically included：Cell is collected in digestion, with sodium chloride injection
It is made final concentration of 1~2 × 106/ ml single cell suspension, draws after 10 μ l trypan blue solutions are mixed with 10 μ l samples and blows and beats
It is even, 10 μ l mixing suspensions are taken, with tally into oblique 45 degree of subscripts in cover glass edge, the action are repeated, by the another of tally
Individual cell is full of with remaining 10 μ l cell suspensions；Tally is lain on microscopical objective table and counted；Single chamber counts thin
Born of the same parents must not be less than 400；The blue dye cells on total cells number ratio of observation；Indigo plant dye cell number should be less than 15%, and Cell viability should be big
In 85%.
Method two, using CASY-TT rapid cellulars analyzer detect that Cell viability should be greater than 85%.
S22, the observation for carrying out by inverted microscope cellular morphology, it is thin that the attached cell of collection should be into fibrous fusiformis
Born of the same parents；
S23, mescenchymal stem cell phenotypic characteristic detection
Sample is arranged by bar code number order, if every part of sample according to detection antibody type the need for take streaming dedicated pipe
The Heavenly Stems and Earthly Branches, have marked Isotype control pipe and antibody pipe respectively；Sample is moved into special streaming pipe, 500g is centrifuged 5 minutes；Abandon supernatant,
1ml PBS are added, are fully mixed, 500g is centrifuged 5 minutes, abandons supernatant, the PBS suspension cells precipitation of proper volume is added；
The μ l cell suspensions of sample 100 are separately added into two pipes；Respective amount antibody is separately added into corresponding pipe and corresponding same
Type contrast agents；Fully mix, put 4 DEG C of refrigerator lucifuges and be incubated 30 minutes.1ml PBS are added, are fully mixed, 500g centrifuges 5 points
Clock；Supernatant is abandoned, 1mL PBSs is then added, fully mixes, 500g is centrifuged 5 minutes；Supernatant is abandoned, appropriate PBS is added,
Fully mix, adjustment cell to suitable concn, then upper machine testing；Directly detected in recovery freeze-stored cell or recovery is frozen if necessary
Deposit cell to be further cultured in detecting after 72h, the result of at least one test point should comply fully with claimed below：Positive antigen：
CD29, CD44, CD73, CD90, CD105 (wherein CD29, CD44 are used as sampling observation index, 1 times/year), positive indication expression rate >=
95%；Negative antigens：CD14, CD19, CD34, CD45, HLA-DR, negative indication expression rate≤2%；
S24, cell final wash supernatant 30ml is taken, according to《Sterility testing S.O.P.》Middle hemoculture instrument detection method
Sterility testing is carried out, testing result should meet regulation；
S25, the μ l of cell suspension 100 are taken, according to《Detection of mycoplasma S.O.P.》Carry out detection of mycoplasma, testing result
Regulation should be met；
S26, endotoxin detection：Cell suspension 0.5ml is taken, according to《Endotoxin examination criteria operation sequence》Detection, should meet
Regulation；
S27, cell suspension is taken, according to《Karyotyping S.O.P.》Karyotyping is carried out, cell should be in chromosome water
It is flat to keep stable；
S28, cell suspension is taken, according to《Genomic methylation examination criteria operation sequence》Genomic methylation detection is carried out,
The methylation level of genome should be unchanged；
S29, cell suspension is taken, according to《Telomerase activation examination criteria operation sequence》Carry out telomerase activation detection, cell
Telomerase activation should be unchanged.
S3, provided (recovery) cell calibrating；Specifically include following steps：
Cell is collected in S31, digestion, and final concentration of 1~2 × 106/ml single cell suspension is made with sodium chloride injection,
Draw and blow and beat uniform after 10 μ l trypan blue solutions are mixed with 10 μ l samples, take 10 μ l mixing suspensions, with tally into oblique 45 degree of angles
Note in cover glass edge, repeat the action, another cell of tally is full of with remaining 10 μ l cell suspensions；It will count
Plate lies on microscopical objective table and counted；Single chamber, which counts cell, must not be less than 400；The blue dye cells on total cells number of observation
Ratio.Indigo plant dye cell number should be less than 15%, and Cell viability should be greater than 85%；
S32, cell final wash supernatant 30ml is taken, according to《Sterility testing S.O.P.》Middle hemoculture instrument detection method
Sterility testing is carried out, as a result should meet regulation；
S33, the cell provided every time, after packing terminates, take remnants cell suspension from (final step) separation container
A little, Gram's staining detection is carried out, should be without Gram-negative bacteria or gram-positive bacteria.
S4, on request completion packaging：Using preceding according to doctor's advice, a number of cell is taken to be suspended in preservation liquid respectively, eventually
Volume and packaging are as follows：
The disposable cell cryopreservation tube packagings of 2ml, 1 ± 0.2ml/ pipes, wear for waist and use；The disposable plastics of 50ml
Blood bag is packed, wherein：It is 30 ± 5ml/ bags, used for intravenous injection for 12 years old patient of ＞；25 ± 3ml/ bags, noted for≤12 years old patient's vein
Penetrate use.
S5, frozen, preserved, transport and the term of validity
- 196 DEG C freeze, and the term of validity is tentative 10 years；6.2 4~10 DEG C of stored refrigerated and transports.The term of validity 12 hours
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations, it is characterised in that comprise the following steps：
S1, a certain amount of bleeding of the umbilicus sample of extraction, carry out six detections of infectious disease；
S2, will pass through six detect after bleeding of the umbilicus sample carry out freeze-stored cell product calibrating；
S3, provided (recovery) cell calibrating；
S4, on request complete pack, freeze, transporting and the term of validity demarcation.
2. a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations as claimed in claim 1, it is characterised in that described
Six detections include HbsAg, anti-HCV, syphilis helicoid antibody, anti-HIV, cytomegalovirus and ALT detection；Wherein preceding four
Person detects that testing result should be negative using ELISA；Cytomegalovirus detected using ELISA, IgG and IgM's
Testing result should be negative；ALT detects that testing result answers≤40 units using performance rate method.
3. a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations as claimed in claim 1, it is characterised in that described
Step S2 specifically includes following steps：
S21, Cell viability detection；
S22, the observation by inverted microscope progress cellular morphology, the attached cell of collection should be into fibrous spindle cell；
S23, mescenchymal stem cell phenotypic characteristic detection
Sample is arranged by bar code number order, every part of sample according to detection antibody type the need for take streaming dedicated pipe some
Branch, has marked Isotype control pipe and antibody pipe respectively；Sample is moved into special streaming pipe, 500g is centrifuged 5 minutes；Supernatant is abandoned, then
1ml PBS are added, are fully mixed, 500g is centrifuged 5 minutes, abandons supernatant, the PBS suspension cells precipitation of proper volume is added；
The μ l cell suspensions of sample 100 are separately added into two pipes；Respective amount antibody and corresponding homotype are separately added into corresponding pipe
Contrast agents；Fully mix, put 4 DEG C of refrigerator lucifuges and be incubated 30 minutes.1ml PBS are added, are fully mixed, 500g is centrifuged 5 minutes；
Supernatant is abandoned, 1mL PBSs is then added, fully mixes, 500g is centrifuged 5 minutes；Supernatant is abandoned, appropriate PBS is added, fully
Mix, adjustment cell to suitable concn, then upper machine testing；Frozen in the directly detection of recovery freeze-stored cell or recovery if necessary thin
Born of the same parents are further cultured in being detected after 72h, and the result of at least one test point should comply fully with claimed below：Positive antigen：CD29,
CD44, CD73, CD90, CD105, positive indication expression rate >=95%；Negative antigens：CD14, CD19, CD34, CD45, HLA-
DR, negative indication expression rate≤2%；
S24, cell final wash supernatant 30ml is taken, according to《Sterility testing S.O.P.》Middle hemoculture instrument detection method is carried out
Sterility testing, testing result should meet regulation；
S25, the μ l of cell suspension 100 are taken, according to《Detection of mycoplasma S.O.P.》Detection of mycoplasma is carried out, testing result should be accorded with
Close regulation；
S26, endotoxin detection：Cell suspension 0.5ml is taken, according to《Endotoxin examination criteria operation sequence》Detection, should meet regulation；
S27, cell suspension is taken, according to《Karyotyping S.O.P.》Karyotyping is carried out, cell should be protected in Chromosome level
It is fixed to keep steady；
S28, cell suspension is taken, according to《Genomic methylation examination criteria operation sequence》Carry out genomic methylation detection, gene
The methylation level of group should be unchanged；
S29, cell suspension is taken, according to《Telomerase activation examination criteria operation sequence》Carry out telomerase activation detection, cell telomere
Enzymatic activity should be unchanged.
4. a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations as claimed in claim 1, it is characterised in that described
Step S3 specifically includes following steps：
Cell is collected in S31, digestion, and final concentration of 1~2 × 106/ml single cell suspension is made with sodium chloride injection, is drawn
10 μ l trypan blue solutions are blown and beaten uniform after being mixed with 10 μ l samples, take 10 μ l mixing suspensions, with tally into oblique 45 degree of subscripts in
Cover glass edge, repeats the action, another cell of tally is full of with remaining 10 μ l cell suspensions；Tally is put down
It is placed on microscopical objective table and counts；Single chamber, which counts cell, must not be less than 400；The blue dye cells on total cells number ratio of observation
Example.Indigo plant dye cell number should be less than 15%, and Cell viability should be greater than 85%；
S32, cell final wash supernatant 30ml is taken, according to《Sterility testing S.O.P.》Middle hemoculture instrument detection method is carried out
Sterility testing, as a result should meet regulation；
S33, the cell provided every time, after packing terminates, take remnants cell suspension a little from final step separation container, enter
The detection of row Gram's staining, should be without Gram-negative bacteria or gram-positive bacteria.
5. a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations as claimed in claim 3, it is characterised in that described
Step S21 uses trypan blue staining, specifically includes following steps：Cell is collected in digestion, is made with sodium chloride injection dense eventually
Spend for 1~2 × 106/ ml single cell suspension, draws after 10 μ l trypan blue solutions are mixed with 10 μ l samples and blows and beats uniformly, take 10 μ
L mixing suspensions, with tally into oblique 45 degree of subscripts in cover glass edge, repeat the action, another cell of tally are used
Remaining 10 μ l cell suspensions are full of；Tally is lain on microscopical objective table and counted；Single chamber, which counts cell, to be lacked
In 400；The blue dye cells on total cells number ratio of observation；Indigo plant dye cell number should be less than 15%, and Cell viability should be greater than 85%.
6. a kind of appraisal procedure of the umbilical cord mesenchymal stem cells quality of the pharmaceutical preparations as claimed in claim 3, it is characterised in that described
Step S21 detects that Cell viability should be greater than 85% using CASY-TT rapid cellulars analyzer.
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Cited By (3)
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| CN112300984A (en) * | 2020-09-21 | 2021-02-02 | 四川大学华西医院 | Medical hUC-MSCs-MVs batch preparation process and quality control |
| CN112342191A (en) * | 2019-08-07 | 2021-02-09 | 路春光 | hMSC production kit development and quality management standard optimization and system establishment |
| CN114134239A (en) * | 2021-11-25 | 2022-03-04 | 广州烨善生物科技有限公司 | Kit for rapidly evaluating mammalian cell quality by PCR (polymerase chain reaction) method and detection method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342191A (en) * | 2019-08-07 | 2021-02-09 | 路春光 | hMSC production kit development and quality management standard optimization and system establishment |
| CN112300984A (en) * | 2020-09-21 | 2021-02-02 | 四川大学华西医院 | Medical hUC-MSCs-MVs batch preparation process and quality control |
| CN114134239A (en) * | 2021-11-25 | 2022-03-04 | 广州烨善生物科技有限公司 | Kit for rapidly evaluating mammalian cell quality by PCR (polymerase chain reaction) method and detection method thereof |
| CN114134239B (en) * | 2021-11-25 | 2023-09-15 | 广州烨善生物科技有限公司 | Kit for rapidly evaluating quality of mammalian cells by PCR method and detection method thereof |
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