CN110237095A - For treating stem cell injection liquid and its preparation and the application of cartilage defect of osteoarthritis - Google Patents
For treating stem cell injection liquid and its preparation and the application of cartilage defect of osteoarthritis Download PDFInfo
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- CN110237095A CN110237095A CN201910448373.8A CN201910448373A CN110237095A CN 110237095 A CN110237095 A CN 110237095A CN 201910448373 A CN201910448373 A CN 201910448373A CN 110237095 A CN110237095 A CN 110237095A
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- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000004353 tibial menisci Anatomy 0.000 description 1
- 229940098465 tincture Drugs 0.000 description 1
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- 230000017423 tissue regeneration Effects 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
Abstract
The present invention provides a kind of stem cell injection liquid for treating cartilage defect of osteoarthritis and its preparation and application.The injection includes the 5th generation human umbilical cord mesenchymal stem cells and liquid vehicle, wherein the 5th generation human umbilical cord mesenchymal stem cells are suspended in liquid vehicle, concentration is 2.0~3.0 × 107A cell/4.5ml~5.0ml liquid vehicle；The liquid vehicle is the mixed liquor of the mixed liquor or physiological saline and autologous platelet rich plasma of physiological saline or autologous platelet rich plasma or physiological saline and human serum albumin；Percent by volume 5~8% shared by human serum albumin；Percent by volume shared by physiological saline is less than or equal to 10%.The injection is administered in such a way that intravenous injection or osteoarticular cavity locally injecting or both combine, it is 1.0~1.5 × 10 that vein, which infuses cell infusion amount,8Cells/ times, it is 2.0~3.0 × 10 that osteoarticular cavity, which injects cell infusion amount,7Cells/ times/mono- knee.The effect that the present invention treats cartilage of osteoarthritis integrated degree is substantially better than traditional hyaluronic acid (HA) treatment means.
Description
Technical field
The invention belongs to biological medicines and field of biotechnology, and in particular to one kind is for treating novel osteoarthritis with cartilage defect
Stem cell injection liquid and its preparation method and application.
Background technique
Knee osteoarthritis (knee osteoarthritis, KOA) is clinically one of the most common type chornic arthritis disease
Disease increases the degenerative disease that disease incidence obviously increases with the age.The pathological manifestations of KOA are that vasa vasorum is reduced under cartilage, soft
The synthesis and decomposition of bone matrix protein are abnormal, eventually lead to articular chondrocytes degeneration apoptosis, articular cartilage defects and inflammation
Infiltrate cartilaginous tissue；Clinical manifestation is the arthralgia slowly developed, swelling, stiff, limitation of activity and joint deformity.With people
Class life-time dilatation and population are gradually aging, and KOA has become serious public health problem, seriously affect personal lifestyle matter
Amount, to bring huge financial burden to personal, family and society.
The means of clinical treatment KOA are broadly divided into No operation and operative treatment at present.Non-operative treatment includes that physics is treated
Method and medicinal treatment (such as anti-inflammatory analgetic and corticosteroids are aided with glycosaminoglycan).Non-surgical therapy treatment for
Early stage KOA has certain inhibition inflammation and lenitive effect, but is difficult to change the abnormal pathologic process on joint structure.In
Advanced stage KOA patient generally requires operative treatment, such as articular cavity rinses cleaning art, orthopedic osteotomy and joint replacement.Preceding two
Person can mitigate clinical symptoms, but there is no damaged tissue repair, and operation itself have it is damaging；The latter's joint prosthesis uses
Restricted lifetime and somewhat expensive.In recent years, being directed to serious OA patient, self or homogenous cartilage tissue block is transplanted, is self soft
The technical applications such as osteocyte transplanting, there is certain curative effect.But it is the limited life span of cartilage cell, limited donor source, different
Body graft immunogenicity causes the problems such as joint fibrosis complication to be based on the above status, it would be highly desirable to study new kneecap joint
Scorching treatment means improve its clinical therapeutic efficacy.
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is careful as the kind with multi-lineage potential
Born of the same parents gradually show its advantage and optimistic prospect in KOA treatment.MSCs has multi-lineage potential, and secretes a variety of nutrition
The factor and regulatory factor.MSCs realizes the therapeutic effect to KOA by two different mechanism: in directly breaking up or induce
The histocyte of derived stem cells differentiation and regeneration substitution damage；Or it by paracrine action, secretes a large amount of anti-apoptotic, inhibit scorching
Disease, immunological regulation and the cell factor for promoting histocyte and revascularization, the regeneration for mitigating tissue damage, promoting injury tissue
It repairs.
In recent years, human umbilical cord mesenchymal stem cells (umbilical cord MSCs, UC-MSCs) are more and more extensive answers
For basis and clinical research.Compared to the MSCs in other sources, UC-MSCs equally has powerful immunological regulation and inflammation suppression
Function processed, differentiation potential is stronger, and without ethical hindrances, materials be easier, rate of amplification faster, have apparent industrialization system
Standby and clinical application advantage.
Summary of the invention
The purpose of the present invention is to provide a kind of human umbilical cord mesenchymal stem cells to treat the application in osteoarthritis drugs,
Lacking one kind in the prior art with alleviation can the safe and efficient technological means for treating osteoarthritis.
In order to solve the above technical problem, the present invention provides technical solution are as follows: it is described a kind of for treating bone joint
The stem cell injection liquid of scorching cartilage defect, it is characterised in that: the injection includes the 5th generation human umbilical cord mesenchymal stem cells and liquid
Solvent, wherein the 5th generation human umbilical cord mesenchymal stem cells are suspended in liquid vehicle, concentration is 2.0~3.0 × 107A cell/
4.5ml~5.0ml liquid vehicle；
The liquid vehicle is the mixed of physiological saline or autologous platelet rich plasma or physiological saline and human serum albumin
Close the mixed liquor of liquid or physiological saline and autologous platelet rich plasma；
When it is the mixed liquor of physiological saline and human serum albumin that liquid matchmaker is molten, mixed solution shared by the human serum albumin
The percent by volume 5~8% of total volume；
When it is the mixed liquor of physiological saline and autologous platelet rich plasma that liquid matchmaker is molten, the shared mixing of the physiological saline
The percent by volume of overall solution volume is less than or equal to 10%.
The further technical solution of the present invention: the injection adds in process for preparation or does not add dexamethasone, when
When adding dexamethasone, additive amount is the every ml injection of 1mg/.
The present invention preferably technical solution: the 5th generation human umbilical cord mesenchymal stem cells are derived from caesarean operation baby's
The high-purity mescenchymal stem cell separated in umbilical cord tissue, which is placed in complete medium, to be cultivated, and passage was expanded to for the 5th generation
Versatility mescenchymal stem cell；Wherein, the complete medium is by serum-free basal medium and medium additives group
At；The serum-free basal medium includes LONZA UltraCULTURE culture medium, DMEM/F12 culture medium, HyClone
Any one in DMEM low sugar fluid nutrient medium；The medium additives are Porcine HGF L-Glutamine and blood
Clear substitute PALL Ultroser G, it is 1~2%, PALL Ultroser G that wherein L-Glutamine, which adds percent by volume,
Addition percent by volume be 2~5%.
The present invention preferably technical solution: the autologous platelet rich plasma is isolated from the self blood of sufferer of acquisition
The platelet rich plasma come.
It is provided by the invention a kind of for treating the preparation method of the stem cell injection liquid of cartilage defect of osteoarthritis,
Be characterized in that the following steps are included:
The culture of (1) the 5th generation human umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cells used in incubation are complete
Full culture medium is made of serum-free basal medium and medium additives；The basal medium includes LONZA
UltraCULTURE culture medium, DMEM/F12 culture medium, any one in HyClone DMEM low sugar fluid nutrient medium；It is described
Medium additives include serum substitute, Porcine HGF；The medium additives are Porcine HGF L-
Glutamine and serum substitute PALL Ultroser G, it is 1~2% that wherein L-Glutamine, which adds percent by volume,
The addition percent by volume of PALL Ultroser G is 2~5%, and specific incubation step is as follows:
A. umbilical cord is acquired: after delivery of baby, conventional disconnected navel, in umbilical cord at 2~3cm of placenta end, with hemostasis clamp
Then firmly umbilical cord is being ligatured at the haemostatic clamp with silk thread, at ligation between haemostatic clamp, with operating scissors by umbilical cord nipping,
The other end of umbilical cord is also ligatured with silk thread, and the umbilical cord length between two ligation points must be greater than 20cm, and umbilical cord is rinsed,
Disinfection is placed in collecting bottle；
B. it separates umbilical cord China Tong Shi glue (Wharton ' s Jelly): umbilical cord is transferred to sterile culture in an aseptic environment
It measures and umbilical cord length and records in ware, then sterilize flushing, and after cutting and removing cord vessels, tear positioned at amnion and blood
Magnificent Tong Shi glue between pipe is put into sterile petri dish, and 5~10ml phosphate buffer washing colloids are added；
C. the magnificent Tong Shi glue of acquisition China's Tong Shi glue (Wharton ' s Jelly) tissue homogenate block preparation: is transferred to centrifugation
It shreds in pipe and repeatedly and 1~4mm is made3Magnificent Tong Shi glue tissue be homogenized block, and colloid homogenate block in umbilical cord mesenchyma is added
Stem cell 10~20ml of complete medium, shakes gently, and is centrifuged 6 minutes under conditions of revolving speed is 1900 turns/min under room temperature,
Reject remainder supernatant, weighing record, according to weight culture；
D. inoculated and cultured primary cell and cell harvest tissue block for the first time: according to gel weight, it is dry that umbilical cord mesenchyma is added
Cell culture complete medium about 0.5g/ml is blown and beaten the colloid homogenate block in step c repeatedly uniformly, by 0.5g/ with electric pipettor
Umbilical cord mesenchymal stem cells complete medium is added in 5-10ml ratio in homogenate, moves to culture according to 15-20ml/ bottles after mixing
In bottle, and tissue homogenate block is made to be uniformly distributed in entire bottom of bottle, is placed in carbonated content 5 ± 0.2%, temperature 37 ± 0.5
DEG C constant temperature and humidity incubator in, the orange cell clone regimental commander of culture solution color under the 12-14 days of originally culture, microscope
To bottom of bottle area 70%-80% when, the primary cell cultivated for the first time is harvested by centrifugation in digestion, and adherent not firm remnants are collected by centrifugation
Tissue block carries out second incubation to remnant tissue's block；
E. tissue block second incubation primary cell and cell harvest: by primary cell, culture bottle number halves calculating for the first time, and
The fresh umbilical cord mesenchymal stem cells complete medium of 10-20ml, the residual tissue block that will be harvested in step d are added in culture bottle
Piping and druming is averagely inoculated in new culture bottle after mixing, and is placed in the constant temperature of carbonated content 5 ± 0.2%, 37 ± 0.5 DEG C of temperature
It is carried out second incubation 5-6 days in constant humidity incubator, until when cell fusion reaches bottom of bottle area 85%~90%, digestion is collected by centrifugation
The primary cell of second incubation, digestion, centrifugation process and step d in for the first time primitive cell culture when digestion, be centrifuged
Cheng Xiangtong；
F. cell passage, amplification: the primary cell that step d and e are collected, with every cm2It is culture medium 5000~6000 thin
The density of born of the same parents is seeded in the constant temperature and humidity incubator for the same condition for being placed in primitive cell culture in new culture vessel and passes
It is commissioned to train feeding, when culture to cell fusion degree is up to 75%~85%, harvests the first generation (P1) stem cell, and successively pass with same isodensity
Generation, repeated amplification culture to the 5th generation；
(2) the 5th generation human umbilical cord mesenchymal stem cells cultivated in step (1) are pressed 2.0~3.0 × 107Cell/4.5~
The amount of 5ml, which is suspended in liquid vehicle, is configured to stem cell injection liquid；Wherein, the liquid vehicle is physiological saline, or self
The mixed liquor or physiological saline of platelet rich plasma or physiological saline and human serum albumin and mixing for autologous platelet rich plasma
Close liquid；When it is the mixed liquor of physiological saline and human serum albumin that liquid matchmaker is molten, percent by volume 5 shared by the human serum albumin
~8%；When the mixed liquor of the molten physiological saline of liquid matchmaker and autologous platelet rich plasma, volume basis shared by the physiological saline
Frequently it is less than or equal to 10%.
The present invention preferably technical solution: the umbilical cord acquired in a step of the step (1) uses 0.5% Iodophor to disappear first
Poison, then sufficiently rinsed with 75% alcohol, then with the punching of 500ml physiological saline or phosphate buffer wash clean；The step (1)
B step in umbilical cord sample washed umbilical cord sample 2~3 times using phosphate buffer after Iodophor or alcohol disinfecting, is added, often
Secondary addition 5~10ml phosphate buffer washs limpider to buffer；Umbilical cord is first laterally cut off, 0.5~1 centimetre is one section,
It is placed in the culture dish of 5~10ml phosphate buffer, then umbilical cord is cut off in longitudinal direction, is cut with sawtooth tweezer and is moved towards by blood vessel spiral
Two arteries of umbilical cord are rejected, a vein adds mucous layer；Then China will be led between amnion and blood vessel with long handle sawtooth tweezer
Family name's glue, which is torn, to be put into sterile petri dish, and 5~10ml phosphate buffer washing colloids are added.
The present invention preferably technical solution: it is characterized in that primary cell is trained for the first time in the step d of the step (1)
Feeding process is specific as follows: at the 4th~6 day of originally culture, culture solution was in light yellow, and naked eyes visible tissue homogenate block becomes
It is small, when micro- red visible 5~10% cell of culture solution color climbs out of under mirror, fresh stem cell complete medium 10ml is supplemented in culture
In the culture bottle of cell, continue to cultivate in the carbon dioxide constant temperature and humidity incubator under the conditions of being placed in together；Originally culture the 8th~
10 days, culture solution be in again it is light yellow, naked eyes visible tissue homogenate block become smaller, culture solution color yellowish visible 20~50% under mirror
Cell climbs out of, and supplements fresh stem cell complete medium 10ml again in the culture bottle of culture cell in the ratio of 1:1, is placed in
With culture is continued in condition carbon dioxide constant temperature and humidity incubator to the 12nd~14 day, culture solution color is orange thin under microscope
Born of the same parents clone regimental commander to bottom of bottle area 70%~80% when, shake gently and pat several lower culture bottles, make adherent not firm tissue
Block falls off；The tissue block to fall off and culture solution are collected together to 50ml centrifuge tube, lower 1900 revs/min of room temperature, are centrifuged 6 points
Clock removes supernatant, collects tissue pieces in centrifuge tube and waits for secondary primitive cell culture；And it is added with 10ml phosphate buffer
It in Tissue Culture Flask, gently rinses culture bottle 1 time, abandons cleaning solution, then 5~10ml pancreatin (rewarming is added into Tissue Culture Flask
To room temperature) it is placed in carbon dioxide constant temperature and humidity incubator and starts vitellophag, Tissue Culture Flask, which is quickly removed, after 3 minutes gently claps
Playing culture bottle makes cell fast-falling get off to be added 5~10ml/ bottles of termination digestion of terminate liquid, collects cell to 50ml centrifuge tube
In, lower 1900 turns of room temperature are centrifuged 6 minutes, remove supernatant, and collection obtains once cultivating primary cell.
The present invention preferably technical solution: during carrying out secondary culture in the f step of the step (1), passage is collected
Culture 1/3~2/3 first generation or 4/5 second generation cell with 5 × 106Cell/ml density suspension is dry thin in serum-free
The first generation (P1), the second generation (P2) master cell bank are built in born of the same parents' frozen stock solution；Remaining stem cell is prepared cell by same isodensity to hang
Liquid is inoculated in culture bottle and continues amplification cultivation；By cultured 5th generation human umbilical cord mesenchymal stem cells with 5~10 × 106
Cell/ml density suspension dispenses in serum-free stem cell cryopreserving liquid, and by suspension according to 2~3.0 × 107Cell/3ml/
Pipe, builds the 5th generation stem cell (P5) working cardial cell library, and the cell of packing is placed in cell cryopreservation box, freezes 12- at -80 DEG C
Long-term preservation in liquid nitrogen container is gone to after 18 hours.
In the above-mentioned preparation method of stem cell injection liquid for treating cartilage defect of osteoarthritis, the step (3)
The preparation method of autologous platelet rich plasma, specifically includes the following steps:
A. to pre-install the autologous peripheral whole blood that the heparin tube of anti-coagulants acquires gonitis sufferer under sterile working, and divide
It is attached in sterile 50ml centrifuge tube；
B. 10min is centrifuged under conditions of revolving speed is 3000rpm to the whole blood in step a, and draws upper layer blood after centrifugation
It starches in the 50ml centrifuge tube of another Zhi Xin, secondary centrifuging 10min under conditions of revolving speed 3000rmp；
C. the upper plasma in step b after secondary centrifuging is discarded, retains required blood plasma dosage, shakes centrifuge tube after standing,
It is resuspended in tube bottom precipitating in remaining blood plasma, as autologous platelet rich plasma.
A kind of stem cell injection for being used to treat cartilage defect of osteoarthritis of above method preparation provided by the invention
The application of liquid, it is characterised in that: by the injection in such a way that intravenous injection or osteoarticular cavity locally injecting or both combine
It is administered；When using intravenous injection, by injection volume be for 22.5ml~25ml/ times, cell infusion amount for 1.0~1.5 ×
108Cells/ times；When being injected using osteoarticular cavity, injection dosage is 4.5ml~5.0ml/ times/mono- knee, and cell infusion amount is
2.0~3.0 × 107Cells/ times/mono- knee.
Compared with prior art, the invention has the beneficial effects that, human umbilical cord mesenchymal stem cells used pass through specific
Free serum culture technical method is prepared.Cell immunogenicity is weak, and microbiology and biology safety are good, proliferative capacity
By force, obtainable cell quantity is more, and cell differentiation is strong.Under this study condition, by between articular cavity locally injecting umbilical cord
Mesenchymal stem cells significantly alleviate gonitis symptom, mitigate articular cartilage damage, promote injury repair；Without apparent adverse reaction
Occur；The effect for treating cartilage of osteoarthritis integrated degree is substantially better than traditional hyaluronic acid (HA) treatment means.
Detailed description of the invention
Fig. 1 and Fig. 2 is the human umbilical cord mesenchymal stem cells morphologic observation of the 5th generation；
Fig. 3 is the 5th generation human umbilical cord mesenchymal stem cells growth curve；
Fig. 4 is the 5th generation human umbilical cord mesenchymal stem cells surface marker analyte detection；
Fig. 5 is P5 for UC-MSCs osteoblast differentiation related gene expression；
Fig. 6 is external evoked osteoblast tissue staining of the P5 for UC-MSCs；
Fig. 7 is that P5 is expressed for UC-MSCs chondroblast differentiation associated gene；
Fig. 8 is external evoked chondroblast tissue staining figure of the P5 for UC-MSCs；
Fig. 9 is Mankin ' s appraisal result of the P5 for UC-MSCs treatment to Wistar rats osteoarthritis cartilaginous tissue；
Figure 10 is that P5 is treated for UC-MSCs to knee osteoarthritis cartilage defect repair pathologic examination result；
Patients with Knee Osteoarthritis P5 is for UC-MSCs left knee joint results of imaging before and after treatment in Figure 11 embodiment 2.
Patients with Knee Osteoarthritis P5 is for UC-MSCs right knee joint results of imaging before and after treatment in Figure 12 embodiment 2.
Specific embodiment
The invention will be further described for embodiment with reference to the accompanying drawing.
Following embodiment and the specific of the human umbilical cord mesenchymal stem cells (UC-MSCs) in experiment are separately cultured step such as
Under:
(1) acquire umbilical cord: after delivery of baby, routinely break navel, need to be according to relevant regulations if you need to acquire Cord blood after the navel that breaks
Cord blood is first acquired, acquisition bleeding of the umbilicus is such as not required to, can first drain bleeding of the umbilicus from incision position or eliminates navel with sterile gauze extruding wiping
Blood；Then umbilical cord is clamped with haemostatic clamp at 2~3cm of placenta end in umbilical cord, is then being ligatured at haemostatic clamp with silk thread
(silk ligature is in the chitin umbilical cord packet in umbilical cord storage and transportation box), at ligation between haemostatic clamp, with operating scissors by umbilical cord
It cuts, the other end of umbilical cord is also ligatured with silk thread, and the umbilical cord length between two ligation points must be greater than 20cm.
(2) umbilical cord sample cleaning treatment: after the completion of umbilical cord acquisition, with the tire of sterile gauze erasing umbilical cord surface (placenta percreta)
The dirts such as rouge, amniotic fluid；Umbilical cord is held with sterility forceps, is first sufficiently rinsed with 75% alcohol, it is then dry with 500ml normal saline flushing
Only；It if umbilical cord sample tissue is seriously polluted, can first be handled with iodophor disinfection before alcohol rinse, and the umbilical cord of acquisition is placed in and is adopted
Collect in bottle, and is placed in conserving case.
(3) it separates umbilical cord China Tong Shi glue (Wharton ' s Jelly): taking out collecting bottle from conserving case, after checking and finding correct,
Sample collection bottle is opened in Biohazard Safety Equipment between cell preparation, with aseptic nipper transfer umbilical cord into 10cm sterile petri dish,
Measurement umbilical cord length simultaneously records (hereafter so there is operation that must strictly observe aseptic principle aseptic condition in Biohazard Safety Equipment
Lower progress！), the preservation liquid of umbilical cord remains in collecting bottle, and takes out 5ml and save liquid as keeping sample and 1 do pathogen microbial biomass
Inspection；After later by umbilical cord sample using the tincture of iodine or alcohol disinfecting, phosphate buffer washing umbilical cord sample 2~3 times is added, every time
Addition 5~10ml phosphate buffer washs limpider to buffer；Umbilical cord is first laterally cut off, about 0.5~1 centimetre is one
Section, is placed in the culture dish of 5~10ml phosphate buffer, then umbilical cord is cut off in longitudinal direction, is cut to walk by blood vessel spiral with sawtooth tweezer
To two arteries for rejecting umbilical cord, a vein adds mucous layer；It then will be between amnion and blood vessel with long handle sawtooth tweezer
Tear is put into sterile petri dish white connective tissue (as China's Tong Shi glue), and 5~10ml phosphate buffer is added and washs glue
Body.
(4) the magnificent Tong Shi glue of acquisition China's Tong Shi glue (Wharton ' s Jelly) tissue homogenate block preparation: is transferred to one
In 50ml centrifuge tube, with 18cm elbow long handle scissors for surgery, tissue is shredded 5~10 minutes repeatedly, 1~4mm is made3Group
Knit homogenate block；And the complete 10~20ml of culture medium of stem cell will be added in colloid, it shakes gently, lower 1900 turns of room temperature/6 points of centrifugation
Clock, reject remainder supernatant, weighing record, according to weight culture；Final wash liquid (at least 5ml) is left and taken at this time as keeping sample 2,
For pathogeny microbiology quality inspection；
(5) primary umbilical cord mesenchymal stem cells are cultivated for the first time:
A. according to gel weight in step (4), the full culture medium 0.5g/5ml of stem cell is added, with electric pipettor (10ml
Wide-bore tip pipette) it blows and beats uniformly repeatedly, in 0.5g/5ml ratio, add the full culture medium of stem cell to enter in homogenate, mixing moves back
In to T175 culture bottle (20ml/ bottles)；The unique identification code of cell, cell algebra and incubation time are indicated on culture vessel
Etc. information (such as mother or baby's name, baby's gender/cell number such as UC0001 (sample number)-W (work library)-M are (main carefully
Born of the same parents library) culture bottle horizontal is evenly distributed as much as possible tissue homogenate block in entire bottom of bottle, is placed in carbonated by -1 (algebra)
Inside constant temperature (37 ± 0.5 DEG C) the constant humidity incubator of (5 ± 0.2%)；
B. half amount fluid infusion for the first time: the 4th~6 day (culture solution is in light yellow) of originally culture, the homogenate of naked eyes visible tissue
Block becomes smaller, and visible 5~10% cell climbs out of (a small amount of attached cell or also non-attached cell) under mirror, and supplement fresh stem cell is complete
Culture medium 10ml is placed in carbon dioxide constant temperature and humidity incubator in the culture bottle of culture cell and continues to cultivate；
C. second of half amount fluid infusion: originally culture the 8th~10 day (culture solution is in light yellow again), naked eyes visible tissue was even
Lumps becomes smaller and smaller, and visible 20~50% cell climbs out of (visible 30% attached cell and 50% non-attached cell) under mirror,
The full culture medium 10ml of fresh stem cell is supplemented in the culture bottle of culture cell, be placed in carbon dioxide constant temperature and humidity incubator after
Continuous culture.
(6) harvest of primary (P0) cell: the 12nd~14 day of originally culture, cell clone regimental commander to bottom of bottle area
When 70%~80%, several lower culture bottles are shaked gently and patted, adherent not firm tissue block is made to fall off；By the tissue block to fall off with
Culture solution is collected together to 50ml centrifuge tube, lower 1900 revs/min of room temperature, is centrifuged 6 minutes, is abandoned supernatant, collects fragment of tissue
To cultivate again；It is added in Tissue Culture Flask with 10ml phosphate buffer, is gently rinsed culture bottle 1 time, abandoned cleaning solution and abandon
Clearly, it 10ml pancreatin is added then into Tissue Culture Flask is placed in carbon dioxide constant temperature and humidity incubator and start vitellophag, after 3 minutes
Quickly removing Tissue Culture Flask and gently patting culture bottle makes cell fast-falling get off to be added terminate liquid 5ml/ bottles of terminations digestion, receives
Collect cell suspension into 50ml centrifuge tube, lower 1900 turns of room temperature are centrifuged 6 minutes；Supernatant is removed, digestion is harvested by centrifugation trains for the first time
Feeding primary cell；The collected cell in centrifuge tube simultaneously carries out second incubation with the residual tissue block being collected by centrifugation for the first time
Cell afterwards is the primary cell；
(7) tissue block second incubation: halve calculating by the culture bottle number of primary cell for the first time, it is complete that fresh stem cell is added
Full culture medium is averagely inoculated in new culture bottle, in culture vessel after mixing the residual tissue block piping and druming collected in step (6)
The unique identification code of upper mark cell, the information such as cell algebra and incubation time carry out second incubation；
(8) primary cell of tissue block second incubation is collected: second incubation 5~6 days, until cell fusion reaches bottom of bottle area
When 85%~90%, the primary cell of tissue block second incubation is collected as follows；
A. the harvest of primary (P0) cell: removing completely not adherent even tissue lumps in the way of in step (6),
The primary stem cell of adherent growth is gently washed 1 time with phosphate buffer 1 0ml later；Digestion enzyme solutions will be warmed to room temperature again
(0.125%~0.25%Trypsin-EDTA) is added in the flushed culture bottle of phosphate buffer, every 175cm2It is added
10ml digests enzyme solutions, digests 3 minutes；
When b. observing that most cells are become round by shuttle shape under inverted microscope, stem cell terminate liquid 5ml is added
Stop digestion, blow and beat bottom of bottle repeatedly and largely fall off to cell, move into 50ml centrifuge tube, with phosphate buffer 1 0ml flushing
Culture bottle wall 1~2 time, is collected in centrifuge tube；
C. after blowing and beating suspension cell with pipette, add the full culture medium of stem cell or phosphate buffer to 40ml, 150 mesh
Sterile strainer filtering collects filtered fluid into 50ml centrifuge tube, and lower 1900 rpms of room temperature, centrifugation is collected secondary after 6 minutes
The primary cell of culture.
(9) primary cell passage, culture expand, build master cell bank:
A. according to centrifuge tube inner cell precipitation capacity (can suitably merge in several centrifuge tubes cell into a centrifuge tube), add
Entering appropriate phosphate buffer and be settled to 30ml, gently blows and beats the dispersion of resuspension cell and mix, sampling counts, and lower 1900 turns of room temperature
It is centrifuged 6 minutes under conditions of per minute, removes supernatant；According to cell count, it is complete that fresh stem cell is added in centrifuge tube
It is 25000 cells/ml suspension that culture medium, which prepares density,；Resuspension cell is blown and beaten, gently with every cm2Culture medium 5000~
The density (20ml/T175 culture bottle) of 6000 cells, which is seeded in new culture vessel, to pass on；It is indicated on culture vessel thin
The unique identification code of born of the same parents, the information such as cell algebra and incubation time, be placed in carbonated (5 ± 0.2%) constant temperature (37 ±
0.5 DEG C) culture in constant humidity incubator, when culture to cell fusion degree is up to 80% or so, by the above-mentioned steps harvest first generation (P1)
Stem cell (record cellular morphology, motility rate double number and doubling time), and according to cell count, by 1/2~2/3 first generation
Stem cell cryopreserving builds the first generation (P1) master cell bank；Remaining stem cell is prepared into cell suspension by same isodensity, is inoculated in training
It supports in bottle and continues amplification cultivation, and take 5ml first generation stem cell suspension as keeping sample 3, do CHARACTERISTICS IDENTIFICATION and pathogeny microbiology
Quality inspection；
When b. cultivating to cell fusion degree up to 80% or so, and the harvest second generation (P2) stem cell (record cellular morphology, it is living
Rate doubles number and doubling time), supernatant is removed, gently washs the primary stem cell of adherent growth 1 time with phosphate buffer 1 0ml；
Digestion enzyme solutions (0.125%~0.25%Trypsin-EDTA) will be warmed to room temperature again, and it is flushed that phosphate buffer is added
In T175 culture bottle, every 175cm210ml is added and digests enzyme solutions, digests 3 minutes, observes major part under inverted microscope
When cell is become round by shuttle shape, stem cell terminate liquid 5ml is added and stops digestion；Piping and druming bottom of bottle is largely de- to cell repeatedly
It falls, moves into 50ml centrifuge tube；Culture bottle wall 1~2 time is rinsed with phosphate buffer 1 0ml, is collected in centrifuge tube；With shifting
After liquid pipe blows and beats suspension cell, add phosphate buffer to 40ml, the sterile strainer filtering of 150 mesh；Collect filtered fluid to 50ml from
In heart pipe, lower 1900 rpms of the pelleted by centrifugation of room temperature removes supernatant after 6 minutes, collects the second generation (P2) stem cell, and
According to cell count, 4/5 second generation stem cell cryopreserving is built into the second generation (P2) master cell bank；By remaining stem cell by same
Density prepares cell suspension, is inoculated in and continues to pass in original culture bottle, and takes 5ml second generation stem cell suspension as keeping sample 4, stays
Do cell CHARACTERISTICS IDENTIFICATION and the quality inspection of pathogen microbiology.
(10) stem cell passage, amplification cultivation, build working cardial cell library: the second generation (P2) harvested in step (9) is dry thin
Born of the same parents are successively passed on same isodensity, repeated amplification culture to the 5th generation, (are gone after then removing completely not adherent homogenate block
The same step 6) of removing method gently washs the primary stem cell of adherent growth 1 time with phosphate buffer 1 0ml；It will warm to room temperature again
It digests enzyme solutions (0.25%Trypsin-EDTA), is added in the flushed T175 culture bottle of phosphate buffer, every 175cm2
10ml is added and digests enzyme solutions, digests 3 minutes, when observing that most cells are become round by shuttle shape under inverted microscope,
Stem cell terminate liquid 5ml is added and stops digestion.Piping and druming bottom of bottle largely falls off to cell repeatedly, moves into 50ml centrifuge tube, with
Phosphate buffer 1 0ml rinses culture bottle wall 1~2 time, collects in centrifuge tube, after blowing and beating suspension cell with pipette, adds phosphorus
Phthalate buffer to 40ml, the sterile strainer filtering of 150 mesh collects filtered fluid into 50ml centrifuge tube, 1900 revolutions per minute under room temperature
After Zhongli's heart 6 minutes, supernatant is removed, collects the 5th generation human umbilical cord mesenchymal stem cells, and count (record cellular morphology, it is living
Rate doubles number and doubling time), by the stem cell of harvest with 5 × 106Cell/ml density suspension is in stem cell cryopreserving liquid；
Then by the 5th generation human umbilical cord mesenchymal stem cells suspension according to 2.5 × 107Cell/3ml/ pipe, builds five generation human umbilical cord mesenchymals
Stem cell (P5 is for UC-MSCs) working cardial cell library, and the cell of packing is placed in cell cryopreservation box, -80 DEG C are stayed overnight (12~18
Hour), go to long-term preservation in liquid nitrogen container.
Keep sample -5: staying the 5th foundry of 1ml to make stem cell suspension, do CHARACTERISTICS IDENTIFICATION and the quality inspection of pathogen microbiology, tumorigenesis
Property and safety analysis, analysis result it is specifically as shown in table 1:
The quality testing of table 1.UC-MSCs and security evaluation criteria and result
Stem cell complete medium used in above-mentioned incubation is added by serum-free basal medium and culture medium
Object composition；The basal medium includes LONZA UltraCULTURE culture medium, DMEM/F12 culture medium, HyClone DMEM
Any one in low sugar fluid nutrient medium, preferred LONZA UltraCULTURE culture medium in embodiment, in basal medium
In be added to the Porcine HGF L-Glutamine and culture medium total volume percent of culture medium total volume percent 1.5%
3% serum substitute PALL Ultroser G.
Present inventor sees for above-mentioned P5 for the cellular morphology of the culture the 3rd day and the 7th day of UC-MSCs
It examines, goes shown in micro- sem observation UC-MSCs form such as Fig. 1 (Day 3) and Fig. 2 (Day7)；Observation UC-MSCs cellular morphology be in
Spindle shape, the arrangement of flowing water shape, adherent growth.
And it is recorded for P5 for counting, doubling time and growth curve in UC-MSCs cell cultivation process, P5 generation
UC-MSCs cell growth curve is as shown in figure 3, P5 is as shown in table 2 for UC-MSCs cell count, doubling time table:
Table 2 is P5 for UC-MSCs cell count, doubling time table
| Multiple holes | Day2 | Day4 | Day6 | Day8 |
| 1 | 2.50 | 7.50 | 32.50 | 70.00 |
| 2 | 1.10 | 14.38 | 39.00 | 92.50 |
| 3 | 1.60 | 11.13 | 21.50 | 85.00 |
| ave | 1.73 | 11.00 | 31.00 | 82.50 |
| SD | 0.71 | 3.44 | 8.85 | 11.46 |
Cell count in the 8th day is taken, calculates the cell Proliferation doubling time are as follows: 25.84 hours.
Present inventor detects for the P5 of above-mentioned culture for UC-MSCs cell surface marker, detection knot
Fruit is as shown in figure 4, inspection result is shown, human umbilical cord mesenchymal stem cells the surface marker CD73, CD90 of this batch cultivation,
CD105 positive expression rate is equal>and 95%, CD34, CD14, CD45, CD19, HLA-DR positive expression rate<2%, meet international thin
Born of the same parents' therapy association (ISCT) mescenchymal stem cell surface marker standard of perfection formulated in 2006, cell uniformity are high.
Experiment 1: present inventor induces for UC-MSCs cell through defined medium for the P5 of above-mentioned culture to be broken up
Culture, detection changes with Osteoblast Differentiation related gene expression amount and specific stain has carried out it is demonstrated experimentally that its specific experiment process
It is as follows: the P5 to be measured of routine culture liquid will be resuspended in after recovery for UC-MSCs cell, by 5.0 × 103Cells/well is seeded to 6 holes
It in culture plate, is cultivated under the conditions of 37 DEG C, 5%CO2, saturated humidity, when cell fusion degree reaches 60~80%, induction group is thin
Born of the same parents are changed to Osteogenic Induction Medium, and control group still uses complete medium, replace culture medium 1 time within every 3 days respectively, co-culture 14
Or 28 days.After culture 14 days, culture medium is removed, PBS is cleaned 1 time, and TRIzol reagent 1mL is added in every hole, repeatedly piping and druming dissolution cell,
It is spare to extract RNA.Remaining induction group and cellular control unit continued culture to the 28th day, and 4% formalin fixes cell
30min, 2% alizarin red S (pH4.2) dye 30min, and PBS is cleaned 3 times.Microscopically observation is taken pictures.Fluorescence quantitative PCR detection
Reverse transcription cDNA synthesis (is operated) by TaKaRa company PrimeScriptTM RT reagent Kit with gDNA specification,
And the expression of characterizing gene (Runx2, OPN, ALP etc.) relevant to cell Osteoblast Differentiation type is detected through fluorescence quantitative PCR method
Situation.
Its osteoblast differentiation related gene expression situation as shown in figure 5, P5 for UC-MSCs osteogenic induction 14 days, fluorescence
Quantitative PCR shows that gene expression conspicuousness relevant to osteoblast differentiation increases.Induction group vs. control group: *: P <
0.05；*: P < 0.01.Its P5 is for UC-MSCs osteogenic induction culture 28 days, alizarin red S dyeing, specifically as shown in fig. 6, in Fig. 6
(A) control group is negative, (B) osteogenic induction group display calcific deposit, and × 200.Pass through the external evoked Osteoinductive differentiation knot of UC-MSCs
Fruit confirms that this batch human umbilical cord mesenchymal stem cells have and induces differentiation into bone ability.
Experiment 2: present inventor induces for UC-MSCs cell through defined medium for the P5 of above-mentioned culture to be broken up
It cultivates, detect and changes at cartilage differentiation related gene expression amount and specific stain has carried out it is demonstrated experimentally that its specific experiment mistake
Journey is as follows: the P5 after collecting recovery is for UC-MSCs cell, with 1 × 107The density of a cell/mL is resuspended in complete culture solution；
Cell suspension is added dropwise to 6 porocyte culture plates bottoms (15uL/ drop, 5~7 drops/hole), induction group and control group are set；Carefully
It moves in cell incubator, after 5%CO2,37 DEG C of CMC model 2h, the hole complete medium 2mL/ is added, continues to cultivate
Night waits for that cell drop is shunk and forms cell microsphere.After cell microsphere is formed, induction group is replaced with chondrocyte induction differential medium, right
Complete medium is continued to use according to group, replaces culture medium 1 time within every 3 days, cultivates 14 or 28 days respectively.After culture 14 days, induction is taken
Group and each 1 hole of cellular control unit, remove culture medium, and PBS is cleaned 1 time, is separately added into the hole TRIzol reagent 1mL/, blown and beaten repeatedly to thin
The dissolution of born of the same parents' microballoon, it is spare to extract RNA.After culture 28 days, induced medium is removed, PBS is cleaned cell microsphere 1 time, 4% formaldehyde
Solution fixes cell 30min；PBS is cleaned 2 times, is done following processing respectively: a) directly being visually observed and take pictures；B) 1% alcian blue
Dye 30min；0.1M HCl is cleaned 3 times, removes remaining dyeing liquor；It visually observes and takes pictures.C) to the cell microsphere after fixation
Routine paraffin wax embedding, film-making, dewaxing and cleaning are carried out, 0.1% safranin O dyeing liquor dyeing 1min is then added, and successively use
The transparent 5min dehydration of 95% 2~3s of ethyl alcohol, dimethylbenzene 15min, dimethylbenzene II, finally fixes mounting with neutral gum mounting liquid,
It takes pictures under the microscope.It operates and synthesizes by TaKaRa company PrimeScriptTM RT reagent Kit with gDNA specification
CDNA, and through fluorescence quantitative PCR method detection to cell at cartilage differentiation type relevant characterizing gene (COL2A1, ACAN, MIA
Deng) expression.
Its chondroblast differentiation associated gene expression as shown in fig. 7, P5 for UC-MSCs osteogenic induction 14 days, it is glimmering
Fluorescent Quantitative PCR shows that gene expression relevant to chondroblast differentiation significantly increases.Induction group vs. control group: *: P <
0.05；*: P < 0.01.And dyed for P5 for the external evoked chondroblast differentiated tissue of UC-MSCs, specifically such as Fig. 8 institute
Show, human umbilical cord mesenchymal stem cells microballoon is at after chondrocyte induction culture 28 days, the white cartilage sample appearance of microballoon (A in such as Fig. 8
It is shown), A Li Xinlan dyeing display cartilage proteoglycan positive staining (as shown in B in Fig. 8), cell microsphere specimens paraffin embedding slices
Safranin O (as shown in C in Fig. 8) and alcian blue dyeing (as shown in D in Fig. 8), above-mentioned colored graph are shown in a large amount of cells in microballoon periphery
The epimatrix component positive colours (× 100).
Embodiment 1: it is used to prepare the stem cell note for the treatment of of arthritis using the human umbilical cord mesenchymal stem cells of above-mentioned culture
Liquid is penetrated, specific process for preparation is as follows:
Washing lotion (DPBS) packing is spare into the centrifuge tube of 15ml；Centrifuge is opened, 1900rpm, 6min are adjusted to；Prepare
Good liquid vehicle: autologous platelet rich plasma, human serum albumin, cell screen, physiological saline；Respective counts are taken out in liquid nitrogen container
The freeze-stored cell of amount is transferred quickly in 37 DEG C of water-baths；(the water bath time when cell cryopreservation tube medium floe residue to soya bean size
1.5~2.5 minutes), cryopreservation tube is taken out, preparation area is transferred quickly to.It is sterilized with sterile alcohol wipe cryopreservation tube, and unscrews lid
Son；Cell is transferred in ready washing lotion with liquid-transfering gun, 1900rpm, is centrifuged 6min.Supernatant is removed after centrifugation, then uses DPBS
It washes 1 time, then is centrifuged supernatant, after cell is filtered with screen, by 2.0~3.0 × 1074.5~5ml liquid is added in a cell/every pipe
Body solvent slowly stirs evenly the stem cell injection liquid for being configured to specification as 5ml, and has prepared the note of following four various combination
Penetrate liquid:
Composition 1:P5 is for UC-MSCs cell+physiological saline
Composition 2:P5 is for UC-MSCs cell+physiological saline+human serum albumin
Composition 3:P5 is for UC-MSCs cell+autologous platelet rich plasma
Composition 4:P5 is for UC-MSCs cell+autologous platelet rich plasma+physiological saline
In above-mentioned four kinds of compositions, when carrying out animal experiment study using physiological saline or physiological saline and the white egg of people's blood
White mixture is as liquid vehicle, when the mixture using physiological saline and human serum albumin is as liquid vehicle, people
Blood albumin is the 5~8% of solvent percent by volume；When carrying out human body therapy, generally made using autologous platelet rich plasma
For liquid vehicle, when autologous platelet rich plasma content is inadequate, physiological saline can be used as compensation, but physiological saline
Additive amount is not more than 10%.
Embodiment 2: this experiment of effectiveness study of knee joint osteoarthritis animal model is selected in fact for UC-MSCs for P5
The cell therapy composition 1 (stem cell+physiological saline) prepared in example 1 is applied, its curative effect to knee osteoarthritis is investigated, it is specific
Experimentation is as follows:
60 healthy adult male Wistars (weight 175g~200g) are selected to be divided into four groups by random digits table: just
Normal group (n=15), model group (n=15), stem-cell therapy group (n=15), HA treatment group (n=15).Normal group is without appointing
The rat of where reason, model group are the knee osteoarthritis model of sodium iodoacetate (Monosodium iodoacetate, MIA) induction
Rat, stem-cell therapy group are the rat that knee joint cavity injects UC-MSCs, and HA treatment group is that knee joint cavity injects HA.After modeling,
14 days after knee osteoarthritis.Cell therapy group and object 1 (contain 1 × 10 in stem-cell therapy group joint cavity injection embodiment 16It is dry thin
The normal saline suspension of born of the same parents) 100 μ L, HA treatment group joint cavity injection, 100 μ L HA, negative control group joint cavity injection 100
μ L physiological saline.Cell infusion treat 4 weeks after, put to death experimental animal, collect Rat Right patella, to articular cartilage surface into
Row gross examination of skeletal muscle and cartilaginous tissue pathological observation, and according to articular cartilage surface gross examination of skeletal muscle standards of grading and Mankin cartilage
Histopathological scores standard scores.T is examined carrying out group to each experimental group appraisal result, and P < 0.05 is that difference has statistics
Learn meaning.
After two weeks, rat knee joints are observed in joint cavity injection UC-MSCs, treatment to kneecap joint cavity injection MIA afterwards three times
Pathological change, it is specific as shown in Figure 9 and Figure 10, staining pathologic section as the result is shown: the edge out-of-flatness of model group articular cartilage, it is soft
Bone thickness and cartilage cell's reduction etc.；And UC-MSCs treatment group, cartilage structure tend to completely, cartilage cell increases, therapeutic effect
Bright group aobvious better than model (solvent) group and HA treatment group.If the Mankin appraisal result of the fast green slice of sarranine-in Figure 10 is shown,
UC-MSCs treatment group cartilage integrated degree is significantly better than model (solvent) group and HA treatment group.Mankin ' s appraisal result is shown
The knee articular cartilage defect of umbilical cord mesenchymal stem cells group treatment significant improvement animal pattern, * P < 0.05, * * P <
0.01, * * *: P < 0.001.
It is treated through UC-MSCs and HA, rat knee joints slice H&E, toluidine blue or the fast green coloration result of sarranine-are shown:
The edge out-of-flatness of model group articular cartilage, thickness are thinning, the de- dye of matrix, and cartilage cell is reduced etc.；The above-mentioned disease of UC-MSCs treatment group
Become apparent improvement, and close to normal group, therapeutic effect is better than hyaluronic acid (HA) treatment group (× 200)；Rat kneecap joint injury
Mankind scoring shows that UC-MSCs treatment group is substantially better than model group and hyaluronic acid (HA) treatment group；Rat kneecap joint kind
Red-fast green stained slice carries out Mankin scoring using blind by three researchers；Mean ± the SD to score as the result is shown, * * *:
P < 0.001, ns: not statistically significant.
Embodiment 3: using the composition 3 (stem cell+autologous platelet rich plasma) prepared in embodiment 1 to knee joint bone
Arthritic clinical application case 1:
Patient basis: patient Zhao so-and-so, male, 51 years old, occupation: police/wushu athlete.Physical examination in November, 2018,
Main suit: double knee joint pain 20+ repeatedly is aggravated 4 years；Present illness history: patient because of double knee joint pain caused by long-term a large amount of movement,
Gradually aggravate, walking, tranquillization when feel double knee joint pain, right side be very.With snap sound, row when movable in the knee joint of right side
The treatment such as acupuncture, locally injecting sodium hyaluronate, has no positive effect, pain gradually aggravates over nearly 4 years, and abnormal with right knee joint
Shape.Since morbidity, spirit, diet can, sleep it is not good enough.Past medical history: exist because of right side injury of meniscus of knee joint in outer court within 2014
Meniscus of knee joint enucleation on the right side of knee arthroscope downlink.
Training inspection: double lower limb is isometric, and double hip joint activity is normal, and right knee is in Varus deformity, and each muscle group of double lower limb has no
Obvious atrophy, double knee joint is without obvious tumefaction, right knee joint 10-0-100, left knee joint 10-0-110；Lateral gap in double knees
Tenderness is obvious, and floating patella test (-), grinding test (-), Lachman test (-), side stress test (-), McMurray sign (+), remaining
It shows no obvious abnormalities.
Tentative diagnosis: 1, double knee osteoarthritis；2, after right medial meniscus of knee joint enucleation；3, double knee joint hydrops
Therapeutic scheme: patient is because double knee joint inflammation is using " the 5th generation human umbilical cord mesenchymal of PRP+ of composition 3 in embodiment 1
The treatment of stem cell " locally injecting, on December 12nd, 2018, patient treats for the first time, acquires self 20~30ml of anticoagulated whole blood, uses
Secondary centrifuging method, 3000rpm/10min extract platelet rich plasma (PRP) for umbilical cord mesenchymal stem cells to be resuspended.
Double knees inject umbilical cord mesenchymal stem cells 2.5 × 10 respectively7A cell；It is carried out by with scheme on March 22nd, 2019
Second for the treatment of.After treating 3 months for the first time, double knee pain improve earlier above, no rest pain, and sleep improves earlier above, walk, squat down,
Double knee joint still has pain when stair activity, and right side knee joint improves earlier above without obvious snap, activity, diet, sleep, spirit,
The ordinary circumstances such as stool and urine can.
Training inspection: double lower limb is isometric, and double hip joint activity is normal.Double knee joint is without swelling, right knee Varus deformity, stock
Atrophy that musculus lateralis interni is slight, right knee joint 10-0-120, left knee joint 5-0-130；Tenderness is obvious at double knee joint " pes anserinus ", floats kneecap
(-), grinding test (-), Lachman test (-), side stress test (-), McMurray sign (+) are tested, and for pretherapy and post-treatment
Left and right knee joint magnetic resonance imaging Comparative result (MRI), it is specific such as Figure 11 (left knee joint contrast before and after treatment) and Figure 12 (right knee
Comparison before and after treatment of joint disease), before treatment: right knee joint 10-0-100, left knee joint 10-0-110；After treatment: right knee joint 10-
0-120, left knee joint 5-0-130.
Embodiment 4: using the composition 3 (stem cell+autologous platelet rich plasma) prepared in embodiment 1 to knee joint bone
Arthritic clinical application case 2:
Patient basis: patient Shen xx, female, 55 years old, occupation: General Office Clerk, consultation time: December 27 in 2018
Day, main suit: double knee joint pain 5 years, patient started double knee joint pain occur in five years ago without obvious inducement, squats down and bears a heavy burden
When pain it is obvious, occasionally double knee joint morning stiffness about 10 minutes, voluntarily can be relieved after activity, nothing with snap when right knee articulation
Rest pain, give acupuncture, locally injecting sodium hyaluronate etc. treatment after, no positive effect, pain still repeatedly, since onset, spirit,
Food intake, sleep can, stool and urine is normal.
Training inspection: double lower limb is isometric, and each muscle group has no obvious atrophy, and double knees have no swelling, double knees without obvious deformity
Kneecap lower edge inside tenderness (+-), double knee joint buckling is slightly limited, right knee mobility 5-0-100 degree, left knee mobility 5-0-
110 degree, limb terminal blood moving and feeling are normal.Double knee kneecap grinding tests (-), APLEY test (+), bilateral McMurray sign (+), drawer
It tests (-), side stress test (-), pivot shift test (-) is remaining to show no obvious abnormalities.
Tentative diagnosis: 1, double knee osteoarthritis；2, left gonyocele；3, Zuo popliteal cyst.
Therapeutic scheme: patient is because double knee joint inflammation is using " the 5th generation human umbilical cord mesenchymal of PRP+ of composition 3 in embodiment 1
The treatment of stem cell " locally injecting, completion on January 7th, 2019 pair knees " double knee stem cell+PRP treatments " for the first time, treatment is specific
It is the self 20~30ml of anticoagulated whole blood of acquisition, using secondary centrifuging method, 3000rpm/10min is extracted platelet rich plasma (PRP)
For umbilical cord mesenchymal stem cells to be resuspended, double knees inject 2.5 × 107 cells of umbilical cord mesenchymal stem cells respectively.Patient tells row
Double knee stem-cell therapy 3 months after operation, it is substantially reduced earlier above to feel double knee pains at present, can walk over long distances and stair activity,
The swollen discomfort of the double knees acid of random thoughts, has apparent therapeutic effect.
Claims (10)
1. a kind of for treating the stem cell injection liquid of cartilage defect of osteoarthritis, it is characterised in that: the injection includes the 5th
For human umbilical cord mesenchymal stem cells and liquid vehicle, wherein the 5th generation human umbilical cord mesenchymal stem cells are suspended in liquid vehicle,
Concentration is 2.0~3.0 × 107A cell/4.5ml~5.0ml liquid vehicle；
The liquid vehicle is the mixed liquor of physiological saline or autologous platelet rich plasma or physiological saline and human serum albumin,
Or the mixed liquor of physiological saline and autologous platelet rich plasma；
When it is the mixed liquor of physiological saline and human serum albumin that liquid matchmaker is molten, percent by volume 5 shared by the human serum albumin
~8%；
When it is the mixed liquor of physiological saline and autologous platelet rich plasma that liquid matchmaker is molten, the physiological saline accounts for percent by volume
Less than or equal to 10%.
2. according to claim 1 a kind of for treating the stem cell injection liquid of cartilage defect of osteoarthritis, feature
Be: the injection adds in process for preparation or does not add dexamethasone, and when adding dexamethasone, additive amount is
The every ml injection of 1mg/.
3. according to claim 1 a kind of for treating the stem cell injection liquid of cartilage defect of osteoarthritis, feature
Be: the 5th generation human umbilical cord mesenchymal stem cells are high-purity by separating in the umbilical cord tissue of caesarean operation baby
Degree mescenchymal stem cell, which is placed in complete medium, to be cultivated, and passage is expanded to the versatility mescenchymal stem cell in the 5th generation；
Wherein, the complete medium is made of serum-free basal medium and medium additives；The serum-free basis culture
Base includes LONZA UltraCULTURE culture medium, DMEM/F12 culture medium, in HyClone DMEM low sugar fluid nutrient medium
Any one；The medium additives are Porcine HGF L-Glutamine and serum substitute PALL Ultroser
G, wherein L-Glutamine add the addition percent by volume that percent by volume is 1~2%, PALL Ultroser G be 2~
5%.
4. according to claim 1 a kind of for treating the stem cell injection liquid of cartilage defect of osteoarthritis, feature
Be: the autologous platelet rich plasma is the platelet rich plasma separated from the self blood of sufferer of acquisition.
5. a kind of for treating the preparation method of the stem cell injection liquid of cartilage defect of osteoarthritis, it is characterised in that including with
Lower step:
The culture of (1) the 5th generation human umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cells used in incubation are trained completely
Feeding base is made of serum-free basal medium and medium additives；The basal medium includes LONZA
UltraCULTURE culture medium, DMEM/F12 culture medium, any one in HyClone DMEM low sugar fluid nutrient medium；It is described
Medium additives include serum substitute, Porcine HGF；The medium additives are Porcine HGF L-
Glutamine and serum substitute PALL Ultroser G, it is 1~2% that wherein L-Glutamine, which adds percent by volume,
The addition percent by volume of PALL Ultroser G is 2~5%, and specific incubation step is as follows:
A. acquire umbilical cord: after delivery of baby, conventional disconnected navel clamps navel with haemostatic clamp in umbilical cord at 2~3cm of placenta end
Then band is being ligatured at the haemostatic clamp with silk thread, at ligation between haemostatic clamp, with operating scissors by umbilical cord nipping, umbilical cord
The other end also ligatured with silk thread, the umbilical cord length between two ligation points must be greater than 20cm, and umbilical cord is rinsed, is sterilized
It is placed in collecting bottle；
B. it separates umbilical cord China Tong Shi glue: umbilical cord being transferred in sterile petri dish in an aseptic environment and measure umbilical cord length and remember
Record, then sterilize flushing, and cut remove cord vessels after, the magnificent Tong Shi glue torn between amnion and blood vessel is put into
In sterile petri dish, 5~10ml phosphate buffer washing colloids are added；
C. China's Tong Shi glue tissue homogenate block preparation: the magnificent Tong Shi glue of acquisition is transferred in centrifuge tube and shred repeatedly be made 1~
4mm3Magnificent Tong Shi glue tissue be homogenized block, and colloid homogenate block in be added umbilical cord mesenchymal stem cells complete medium 10~
20ml is shaked gently, and is centrifuged 6 minutes under conditions of revolving speed is 1900 turns/min under room temperature, reject remainder supernatant, weighing note
Record, according to weight culture；
D. inoculated and cultured primary cell and cell harvest tissue block for the first time: according to gel weight, umbilical cord mesenchymal stem cells are added
Complete medium about 0.5g/ml is blown and beaten the colloid homogenate block in step c repeatedly uniformly, by 0.5g/5- with electric pipettor
Umbilical cord mesenchymal stem cells complete medium is added in 10ml ratio in homogenate, moves to culture bottle according to 15-20ml/ bottles after mixing
It is interior, and tissue homogenate block is made to be uniformly distributed in entire bottom of bottle, it is placed in 37 ± 0.5 DEG C of carbonated content 5 ± 0.2%, temperature
Constant temperature and humidity incubator in, the orange cell clone regimental commander of culture solution color is extremely under the 12-14 days of originally culture, microscope
When the 70%-80% of bottom of bottle area, the primary cell cultivated for the first time is harvested by centrifugation in digestion, is collected by centrifugation adherent not firm remaining group
Block is knitted, second incubation is carried out to remnant tissue's block；
E. tissue block second incubation primary cell and cell harvest: by primary cell, culture bottle number halves calculating for the first time, and is training
It supports and the fresh umbilical cord mesenchymal stem cells complete medium of 10-20ml is added in bottle, the residual tissue block harvested in step d is blown
It beats after mixing and is averagely inoculated in new culture bottle, it is permanent to be placed in carbonated content 5 ± 0.2%, 37 ± 0.5 DEG C of temperature of constant temperature
It is carried out second incubation 5-6 days in wet incubator, until when cell fusion reaches bottom of bottle area 85%~90%, digestion is collected by centrifugation two
The primary cell of secondary culture；
F. cell passage, amplification: the primary cell that step d and e are collected, with every cm25000~6000 cells of culture medium it is close
Degree, which is seeded in the constant temperature and humidity incubator for the same condition for being placed in primitive cell culture in new culture vessel, carries out secondary culture,
When culture is to cell fusion degree up to 75%~85%, the first generation (P1) stem cell is harvested, and successively pass on same isodensity, repeatedly
Amplification cultivation is to the 5th generation；
(2) the 5th generation human umbilical cord mesenchymal stem cells cultivated in step (1) are pressed 2.0~3.0 × 107Cell/4.5~5ml
Amount, which is suspended in liquid vehicle, is configured to stem cell injection liquid；Wherein, the liquid vehicle is physiological saline, or rich blood is small self
The mixed liquor or physiological saline of plate blood plasma or physiological saline and human serum albumin and the mixed liquor of autologous platelet rich plasma；When
When the molten mixed liquor for physiological saline and human serum albumin of liquid matchmaker, percent by volume 5~8% shared by the human serum albumin；
When the mixed liquor of the molten physiological saline of liquid matchmaker and autologous platelet rich plasma, percent by volume shared by the physiological saline is less than
Or it is equal to 10%.
6. according to claim 5 a kind of for treating the preparation side of the stem cell injection liquid of cartilage defect of osteoarthritis
Method, it is characterised in that: the umbilical cord acquired in a step of the step (1) uses 0.5% iodophor disinfection first, then with 75% alcohol
It sufficiently rinses, then with the punching of 500ml physiological saline or phosphate buffer wash clean；By umbilical cord in the b step of the step (1)
After sample is using Iodophor or alcohol disinfecting, phosphate buffer is added and washs umbilical cord sample 2~3 times, 5~10ml phosphorus is added every time
Phthalate buffer washs limpider to buffer；Umbilical cord is first laterally cut off, 0.5~1 centimetre is one section, has been placed in 5~10ml
In the culture dish of phosphate buffer, then it is longitudinal cut off umbilical cord, cut with sawtooth tweezer and move towards two of rejecting umbilical cord by blood vessel spiral
Artery, a vein add mucous layer, then China's Tong Shi glue will be torn between amnion and blood vessel with long handle sawtooth tweezer and be put into nothing
In bacterium culture dish, 5~10ml phosphate buffer washing colloids are added.
7. according to claim 5 a kind of for treating the preparation side of the stem cell injection liquid of cartilage defect of osteoarthritis
Method, it is characterised in that the process of primitive cell culture is specific as follows for the first time in the step d of the step (1): in primary training
Feeding the 4th~6 day, culture solution is in light yellow, and naked eyes visible tissue homogenate block becomes smaller, and culture solution color is micro- red visible under mirror
When 5~10% cells climb out of, supplement fresh stem cell complete medium 10ml is placed in same condition in the culture bottle of culture cell
Under carbon dioxide constant temperature and humidity incubator in continue to cultivate；At originally culture the 8th~10 day, culture solution be in again it is light yellow,
Naked eyes visible tissue homogenate block become smaller, yellowish visible 20~50% cell of culture solution color climbs out of under mirror, in 1:1 ratio again
Fresh stem cell complete medium 10ml is supplemented in the culture bottle of culture cell, is placed in same condition carbon dioxide constant temperature and humidity
Continue culture in incubator to the 12nd~14 day, the orange cell clone regimental commander of culture solution color is to bottom of bottle area under microscope
When 70%~80%, several lower culture bottles are shaked gently and patted, adherent not firm tissue block is made to fall off；By the tissue block to fall off with
Culture solution is collected together to 50ml centrifuge tube, lower 1900 revs/min of room temperature, is centrifuged 6 minutes, is removed supernatant, collects centrifuge tube
Middle tissue pieces wait for secondary primitive cell culture；It is added in Tissue Culture Flask with 10ml phosphate buffer, gently rinses culture
Bottle 1 time abandons cleaning solution, then 5~10ml pancreatin is added into Tissue Culture Flask and is placed in carbon dioxide constant temperature and humidity incubator and starts
Vitellophag, Tissue Culture Flask is quickly removed after 3 minutes gently patting culture bottle makes cell fast-falling get off to be added terminate liquid 5
~10ml/ bottles of termination digestion, collects cell into 50ml centrifuge tube, and lower 1900 turns of room temperature are centrifuged 6 minutes, removes supernatant, receives
Collection obtains once cultivating primary cell.
8. according to claim 5 a kind of for treating the preparation side of the stem cell injection liquid of cartilage defect of osteoarthritis
Method, it is characterised in that: during carrying out secondary culture in the f step of the step (1), collect the 1/3~2/3 of secondary culture
The first generation or 4/5 second generation cell with 5 × 106Cell/ml density suspension builds first in serum-free stem cell cryopreserving liquid
Generation, second generation master cell bank；Remaining stem cell is prepared into cell suspension by same isodensity, is inoculated in and continues to expand in culture bottle
Culture；By cultured 5th generation human umbilical cord mesenchymal stem cells with 5~10 × 106Cell/ml density suspension is in serum-free
In stem cell cryopreserving liquid, and suspension is dispensed according to 2~3.0 × 107Cell/3ml/ pipe, builds the 5th generation stem cell working cardial cell
Library, and the cell of packing is placed in cell cryopreservation box, long-term preservation in liquid nitrogen container is gone to after freezing at -80 DEG C 12-18 hours.
9. according to claim 5 a kind of for treating the preparation side of the stem cell injection liquid of cartilage defect of osteoarthritis
Method, it is characterised in that the preparation method of step (3) autologous platelet rich plasma, specifically includes the following steps:
A. the autologous peripheral whole blood of gonitis sufferer is acquired under sterile working with the heparin tube for pre-installing anti-coagulants, and be dispensed into
In sterile 50ml centrifuge tube；
B. to the whole blood in step a revolving speed be 3000rpm under conditions of be centrifuged 10min, and draw centrifugation after upper plasma in
In the 50ml centrifuge tube of another Zhi Xin, secondary centrifuging 10min under conditions of revolving speed 3000rmp；
C. the upper plasma in step b after secondary centrifuging is discarded, retains required blood plasma dosage, shakes centrifuge tube after standing, make pipe
Bottom precipitating is resuspended in remaining blood plasma, as autologous platelet rich plasma.
10. for treating the stem cell injection of cartilage defect of osteoarthritis described in a kind of any one of claim 1 to 5
The application of liquid, it is characterised in that: by the injection in such a way that intravenous injection or osteoarticular cavity locally injecting or both combine
It is administered；Be 22.5ml~25ml/ time by injection volume when using intravenous injection, cell infusion amount for 1.0~1.5 ×
108Cells/ times；When being injected using osteoarticular cavity, injection dosage is 4.5ml~5.0ml/ times/mono- knee, and cell infusion amount is
2.0~3.0 × 107Cells/ times/mono- knee.
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