CN110193025A - The excretion body freeze-dried powder in human adipose-derived stem cell source and its preparation method and application - Google Patents
The excretion body freeze-dried powder in human adipose-derived stem cell source and its preparation method and application Download PDFInfo
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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
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Inorganic Chemistry (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Zoology (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of lyophilized preparation of the excretion body in human stem cell source, and the lyophilized preparation includes the excretion body and trehalose in human stem cell, especially human adipose-derived stem cell source.After long term storage, the measured amount of excretion body protein is able to maintain constant lyophilized preparation of the invention.The lyophilized preparation of the excretion body in fat stem cell source of the invention is easy to transport and long term storage；And to organism safe, provided a great convenience to be saved for excretion body from now on application.
Description
Technical field
The present invention relates to stem cell and biomedicine fields.Specifically, the present invention relates to human adipose-derived stem cells
It the freeze-dried powder of excretion body in the source (Human-Adipose Derived Stem Cell, hADSC) and preparation method thereof and answers
With.
Background technique
Fat stem cell is a kind of adult stem cell with the of self-replication capacity and multi-lineage potential, is had immune
A variety of effects such as inhibition, anti-inflammatory and promotion proliferation, show aobvious in a variety of disease models such as heart, liver, kidney, brain
The curative effect of work.Fat stem cell can secrete excretion body by paracrine approach, and information transfering action is played between Various Tissues.
Excretion body is the lipid bilayer structure that a kind of size of cell active secretion is uniform, diameter is 50-200nm
Vesica can be discharged by a variety of different type cells such as mescenchymal stem cell, tumour cell, dendritic cells, fibroblast.Outside
It secretes in body comprising substances such as a large amount of protein, RNA and DNA, plays an important role to cell-tocell exchange.Fat is dry thin
Extracellular body of secreting possesses the biological effects such as immunosupress similar with fat stem cell, anti-inflammatory.Since excretion body is small in size, peace
The advantages such as Quan Xinggao, the graft application of stem cell excretion body is in the Various Tissues such as heart, liver, kidney, brain field.
With the development in excretion body field, the safe preservation of excretion body is extremely important.Excretion body preservation side traditional at present
Method is directly to freeze under the conditions of -80 DEG C.Excretion body of short duration can save in a cold or frozen state, but with the extension of holding time,
The bimolecular film of excretion body is gradually crushed, and activity is significantly lost, it is difficult to for experiment and clinical application.
Therefore, one kind is badly in need of in this field can stablize the excretion body for saving stem cell, especially fat stem cell source
Method.
Summary of the invention
It can stablize the purpose of the present invention is to provide one kind and save the outer of stem cell, especially fat stem cell source
Secrete the lyophilized preparation of the technological means of body and the fat stem cell source excretion body of acquisition, such as freeze-dried powder.
In a first aspect, the present invention provides a kind of lyophilized preparation of the excretion body in human stem cell source, the lyophilized preparation
Excretion body and trehalose comprising human stem cell source.
In a preferred embodiment, the lyophilized preparation is freeze-dried powder.
In a preferred embodiment, the human stem cell is human adipose-derived stem cell.
In a particular embodiment, the lyophilized preparation is obtained by the following method:
1) solution of the solution of excretion body and trehalose is mixed to get to the mixed solution of the two；
2) the resulting mixed solution of step 1) is lyophilized, to obtain the lyophilized preparation.
In a preferred embodiment, the solution of the excretion body mixes in equal volume with the solution of trehalose.
In a preferred embodiment, the solution of the excretion body is mixed with the solution of trehalose, preferably isometric mixing
Afterwards, the final concentration of 10wt% of trehalose.
In a preferred embodiment, the freeze-drying of step 2) can be the jelly of field of biotechnology or chemical field routine
Drying method.
In a particular embodiment, compared to the original excretion body protein measured amount before storage, the lyophilized preparation exists
At room temperature, such as at 25-25 DEG C store 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or
After 12 months, excretion body protein measured amount is higher than 95%, 96%, 97%, 98%, even higher than 99%.
In second aspect, the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition includes described in first aspect
Lyophilized preparation and optional pharmaceutically acceptable excipient.
In the third aspect, the present invention provides a kind of rehydration object of the lyophilized preparation of the excretion body in human stem cell source,
It is characterized in that, the lyophilized preparation of the excretion body in the human stem cell source is lyophilized preparation described in first aspect.
In a preferred embodiment, the rehydration object 5.6-6.3%, preferably 5.9-6.2%, most preferably 6.0%
Hydroxyethyl starch solution, preferably at 37 DEG C be made.
In fourth aspect, the present invention provide it is a kind of treat with kit, the kit is equipped with:
1) lyophilized preparation described in first aspect or pharmaceutical composition as claimed in claim 4；
2) 5.6-6.3%, preferably 5.9-6.2%, most preferably 6.0% hydroxyethyl starch solution；With
3) using the hydroxyethyl starch solution described in 2) make 1) described in lyophilized preparation or pharmaceutical composition rehydration simultaneously
Obtained rehydration object is given to the operation instructions of the object of this needs.
At the 5th aspect, the present invention provides the lyophilized preparation of the excretion body in human stem cell source described in first aspect
Preparation method the described method comprises the following steps:
1) solution of the solution of excretion body and trehalose is mixed to get to the mixed solution of the two；
2) the resulting mixed solution of step 1) is lyophilized, to obtain the lyophilized preparation.
In a preferred embodiment, the solution of the excretion body mixes in equal volume with the solution of trehalose.
In a preferred embodiment, the solution of the excretion body is mixed with the solution of trehalose, preferably isometric mixing
Afterwards, the final concentration of 10wt% of trehalose.
In a preferred embodiment, the freeze-drying of step 2) can be the jelly of field of biotechnology or chemical field routine
Drying method.
In a preferred embodiment, the human stem cell is human adipose-derived stem cell.
In a preferred embodiment, the lyophilized preparation is freeze-dried powder.
At the 6th aspect, the present invention provides the lyophilized preparation of the excretion body in human stem cell source described in the third aspect
The preparation method of rehydration object, which is characterized in that use 5.6-6.3%, preferably 5.9-6.2%, most preferably 6.0% ethoxy
Starch solution, the lyophilized preparation of the excretion body in human stem cell source described in rehydration first aspect preferably at 37 DEG C, thus
Prepare the rehydration object.
The 7th aspect, the present invention provide first aspect described in human stem cell source excretion body lyophilized preparation or
The purposes of the rehydration object of the lyophilized preparation of the excretion body in human stem cell source described in the third aspect in medicine preparation.
In a particular embodiment, the drug is treatment acute hepatic failure, liver fibrosis, cirrhosis, preferably acute
The drug of hepatic failure.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited,
Not repeated them here.
Detailed description of the invention
Fig. 1 shows the cellular morphology of the hASC and hASC freeze-dried powder frozen.
Fig. 2 compares the BCA-OD value of excretion body and excretion body freeze-dried powder.
Fig. 3 shows the NTA particle diameter distribution of excretion body and excretion body freeze-dried powder.
Fig. 4 shows hemolytic test data.
Fig. 5 shows rat active systemic anaphylaxis test result.
Fig. 6 shows that models of passive skin irritability of rats tests skin picture.
Fig. 7 shows models of passive skin irritability of rats test data.
Specific embodiment
Inventor after extensive and in-depth study, it was unexpectedly found that by the excretion body in fat stem cell source with
Lyophilized preparation, which is made, in trehalose together can steadily save the excretion body in fat stem cell source；Especially fat stem cell
The excretion body in the source manufactured excretion body protein measured amount of lyophilized preparation after long-term preservation together with trehalose is constant, from
And excretion body freeze drying technology of the invention is more suitable for long-term preservation.The present invention is completed on this basis.
Fat stem cell
Fat stem cell (adipose-derived stem cells, ADSC) is isolated from adipose tissue
A kind of stem cell with multi-lineage potential.It is derived from a kind of mescenchymal stem cell of adipose tissue, can be divided into
Interstitial class cell, such as osteocyte, cartilage cell or fat cell.ADSC cell can in vitro it is stable proliferation and decline rate
It is low, while there are materials to be easy, organize that a large amount of stem cells can be obtained on a small quantity, it is suitable for large-scale culture, it is small etc. to body injury
Advantage, and its is from a wealth of sources, cylinder storage amount is big, is suitable for autotransplantation, be increasingly becoming research hotspot new in recent years it
One.In a preferred embodiment, fat stem cell as described herein refers to human adipose-derived stem cells.
In recent years, pluripotency human adipose-derived stem cells (hADSC) have become the treatment some serious diseases of the mankind, such as spinal cord
The good cell origin of damage, cerebral apoplexy, acute hepatic failure etc..
In order to illustrate and convenient purpose, the embodiment of the present invention part is from the adipose tissue that human body liposuction obtains
It separates human adipose-derived stem cells (hADSC).But it is known in the art that various sources, including but not limited to commercial source can be passed through
It obtains human adipose-derived stem cells (hADSC).
Excretion body
" excretion body " as described herein has the normally understood meaning of those of ordinary skill in the art, refers to living cells point
The vesicles with lipid bilayer structure secreted.This excretion body has different marker proteins in its surface expression.
Stem cell excretion body can participate in immunological regulation, mitigate inflammation by carrying the protein largely packed and RNA etc.
Disease reaction, promotion angiogenesis, promotion damaged tissues proliferation and Apoptosis etc..It, can due to having inherent targeting characteristic
With by it protein and RNA be delivered directly in recipient cell；And it since it is natural cellular products, can keep away
From the phagocytosis or degradation of body macrophage, access and lysosomal degradation intracellular are avoided, thus when recycling longer in vivo
Between.In addition, excretion body can also penetrate the blood-brain barrier that many drugs are difficult to penetrate.Therefore, hASC excretion body can be from external
Separation is delivered to and is treated in vivo for disease.
The freeze-drying of excretion body with freeze
With going deep into for stem cell excretion body research, the storage-stable of excretion body is also at technical problem urgently to be resolved.
Excretion body is more than saving under the conditions of -80 DEG C at present, therefore, " freezing " as described herein be excretion body is stored directly in it is low
Under temperature, such as in -80 DEG C of refrigerator.But with the extension of holding time, most of excretion cognition is gradually crushed, and activity is substantially
Degree decline, seriously affects experiment and clinical application.
" freeze drying technology " i.e. as described herein " freeze-drying " is exactly that the substance containing large quantity of moisture is dropped in advance
Temperature is frozen into solid, so that solid water is directly distilled under conditions of vacuum and come out, and substance itself is left when freezing
In ice shelf, entire drying carries out at a lower temperature.Therefore, it is freeze-dried special for the substance of many thermal sensitivity
It is applicable in.And freeze-dried powder long-term preservation, performance can be stablized at normal temperature as a kind of solid dry powder doses, infusion is convenient, necessary expense
With cheap.
However, biomembrane be completely excretion body freeze drying technology biggest obstacle, since low gently dried has biomembrane
Compared with macrolesion, inevitably there is serious film Fragmentation Phenomena in excretion body in freeze-drying process, lead to active decline.
The present inventor by the excretion body in human stem cell source be made together with trehalose lyophilized preparation be solved perfectly it is above-mentioned
Problem.For example, the solution of the solution of excretion body and trehalose can will be mixed to get to the mixed solution of the two；It will obtain again
Mixed solution freeze-drying, to obtain the lyophilized preparation.The freeze drying process wherein used can be field of biotechnology or
The freeze drying process of chemical field routine.
Lyophilized preparation of the invention is highly stable, compared to the original excretion body protein measured amount before storage, the freeze-drying
Preparation at room temperature, such as stores 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 at 15-25 DEG C
A month or after 12 months, excretion body protein measured amount was higher than 95%, 96%, 97%, 98%, even higher than 99%.
On the basis of the lyophilized preparation of the excretion body in human stem cell source of the invention, the present invention also provides include institute
The pharmaceutical composition of lyophilized preparation and pharmaceutically acceptable excipient is stated, or kit is used in treatment.
Lyophilized preparation of the invention can carry out rehydration, the preferred hydroxyethyl starch solution of rehydration solution used, example
Such as 5.6-6.3%, preferably 5.9-6.2%, most preferably 6.0% hydroxyethyl starch solution.
Pharmaceutical composition or drug comprising lyophilized preparation of the invention can be used for treating, such as acute hepatic failure, liver
Fibrosis, cirrhosis, preferably acute hepatic failure.
Main advantages of the present invention include:
1. the lyophilized preparation of the excretion body in fat stem cell source of the invention is stablized；
2. the lyophilized preparation of the excretion body in fat stem cell source of the invention is easy to transport and long term storage；With 3.
The lyophilized preparation of the excretion body in the fat stem cell source of invention is to organism safe, to save and answer for excretion body from now on
With providing a great convenience.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine
Condition such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
1. material and method
1.1 material
1.1.1 experiment reagent
0.15% clostridiopetidase A I (Gibco, the U.S.)；DMEM/F12 culture medium (Hyclone, the U.S.)；Fetal calf serum
(Gibco, the U.S.)；0.25% trypsase (Gibco, the U.S.)；BCA kit (Solarbio, China)；Trehalose (state
Medicine, China)；Hydroxyethyl starch (Aladdin)；Human serum albumin (An Boling).
1.1.2 laboratory apparatus
Constant incubator (Thermo, the U.S.)；Centrifuge (TD25-WS, the limited public affairs of Shanghai Lu Xiangyi centrifuge instrument
Department)；Low temperature ultracentrifuge (Eppendorf, Germany)；Particle size analyzer (NS300, Britain)；Superclean bench (the safe and sound sky in Suzhou
Gas Technology Co., Ltd.)；Centrifuge tube (Merk, Ireland) is concentrated by ultrafiltration；Freeze dryer (CHRIST).
1.2 experimental method
1.2.1 the separation and culture of human adipose-derived stem cell
Liposuction takes healthy human body neck fat tissue (signed informed consent agreement), and PBS is cleaned 3 times, is added
0.1% clostridiopetidase A I is placed in centrifuge in 37 DEG C of water-bath digestion 30min, and it is heavy to leave and take cell after 1200r/min centrifugation 5min
It forms sediment, supernatant is abandoned with above-mentioned pelleted by centrifugation again after being resuspended with appropriate PBS, being added containing volume fraction is 10% fetal calf serum
DMEM/F12 culture medium, is inoculated in 10cm culture dish, and cell concentration is 1 × 106/ mL is placed in 37 DEG C, volume fraction 5%CO2
In saturated humidity incubator, liquid is changed after 48h, when observation cell grows to 80%, is passed after the digestion of 0.25% pancreatin is added by 1:3
It is commissioned to train feeding, culture to the 3rd generation is left and taken subsequent experimental and used.
1.2.2 the extraction of human adipose-derived stem cell excretion body
The 3rd generation human adipose-derived stem cell is taken, first with the DMEM/F12 culture medium for containing volume fraction being 10% fetal calf serum
Culture, the basal medium for using serum-free instead when it grows to 80% or more continue culture for 24 hours, and incubation is at 37 DEG C, body
Fraction is 5%CO2Constant temperature incubator in carry out；Supernatant is collected into centrifuge tube afterwards for 24 hours, 0.22 μm of filter filtering
It is transferred in the ultrafiltration concentration centrifuge tube of 100kd afterwards, 40min is centrifuged with 4000 × g under the conditions of 4 DEG C, obtained rich in excretion body
Concentrate is placed in -80 DEG C of refrigerators and stores for future use.
1.2.3 human adipose-derived stem cell and the freeze-drying of excretion body and rehydration
1.2.3.1 human adipose-derived stem cell freeze-drying and rehydration
Human adipose-derived stem cell is digested and is centrifuged, cell is made with 0.8M Trehalose Loading liquid with volume ratio 1:3 in cell precipitation
Suspension is incubated for 6 hours in 37 DEG C, and cell suspension and freeze drying protectant (20% human serum albumin) mix in equal volume, 37 DEG C of balances
30min is placed in freezing storing box in -80 DEG C of pre-freezes for 24 hours.- 50 DEG C of freeze dryer condenser temperature, vacuum pressure are less than 10mbar, freezing
It is dried 24 hours, is stored in 4 DEG C of refrigerators.Rehydration is quickly shaken molten with 6% hydroxyethyl starch solution that temperature is 37 DEG C
Solve freeze-dried powder.
1.2.3.2 the freeze-drying of excretion body and rehydration
Excretion liquid solution mixes in equal volume with aqueous trehalose, makes the final concentration of 10wt% of trehalose, 37 DEG C of balances
30min is placed on refrigerator processing for 24 hours in -80 DEG C of pre-freeze 12h, and -50 DEG C of freeze dryer condenser temperature, vacuum pressure is less than
10mbar freeze-drying process 24 hours, is stored in 4 DEG C of refrigerators.Rehydration temperature is that 37 DEG C of 6% hydroxyethyl starch is molten
Liquid quickly shakes dissolved freeze-dried powder.
1.2.4 human adipose-derived stem cell freeze-dried powder morphology and Flow cytometry
Fat stem cell and fat stem cell freeze-dried powder are placed under microscope respectively, observe two groups of cell quantities and shape
State difference.Human adipose-derived stem cell freeze-dried powder rehydration cell is taken, 7 pipe 500ul single cell suspensions are made, are separately added into anti-human
CD90-FITC, CD44-PE, CD105-PerCP, CD73-APC, nothing, negative antibody population, each 2ul of positive antibody group, 37 DEG C incubate
It is cleaned 3 times after educating 30min with PBS, uses flow cytomery.
1.2.5 excretion body freeze-dried powder detects
1.2.5.1 excretion body freeze-dried powder BCA is detected
Excretion body is divided into two groups of equivalent, one group of freeze-dried rehydration, another group is not done specially treated, respectively by two groups
Excretion body carries out the detection of BCA protein content, explores excretion body protein content difference.
1.2.5.2 excretion body freeze-dried powder NTA is detected
1mL suitable concentration solution is respectively prepared in excretion body and excretion body freeze-dried powder, utilizes Nanosight NS300 particle
Analyzer testing result excretion body sample powder, injects a sample into sample powder tank, and until filling, adjustment software parameter keeps video clear, point
The RUN key in software is opened, data are collected.
1.2.5.3 excretion body freeze-dried powder transmission electron microscope detects
1 about 10 μ g excretion bodies of drop of drop or excretion body freeze-dried powder take 1 copper mesh to cover in liquid on sealed membrane with tweezers respectively
Then 5-10min in drop drips 1 2% phosphotungstic acid dye liquor of drop on sealed membrane, copper mesh is moved on to phosphorus from excretion body fluid drop with tweezers
On wolframic acid drop, 3min is dyed, liquid is blotted with filter paper, is put under incandescent lamp and dries, with transmission electron microscope observing and take pictures.
1.2.6 excretion body freeze-dried powder safety detection
1.2.6.1 vascular stimulation tests
Take 2kg male rabbit 18, be randomly divided into 6 groups, respectively EXO freeze-dried powder group, EXO group, hASC freeze-dried powder group,
HASC group, physiological saline group, BSA positive controls (5mgBSA), rabbit two sides ear vein dropleting medicine-feeding 10ml, daily 1
Secondary, for three days on end, nearby whether there is or not redness, scabs etc. at observation injection.In last dose for 24 hours afterwards by rabbit sacrificed by exsanguination, ears are taken,
12h is impregnated with 4% paraformaldehyde solution, at injection site proximal part 1cm, venous vascular tissue, longitudinal malingering are taken out in dissection
Reason slice, HE dyeing.Rabbit blood vessel anaphylaxis evaluation criterion are as follows: 0 grade, medicine-feeding part is without significant reaction；1 grade, medicine-feeding part is quiet
Arteries and veins blood vessel is slight hemostasis, redness；2 grades, medicine-feeding part vein blood vessel moderate hemostasis, redness；3 grades, medicine-feeding part vein blood vessel
Severe hemostasis, surrounding tissue are seriously red and swollen.Pathological examination index: the integrity degree of vein endothelial cell, whether there is or not it is congested,
Denaturation, necrosis change, and whether basilar memebrane is complete, the complete situation etc. of tube wall and pipe week tissue.
1.2.6.2 muscle irritation is tested
Take 2kg male rabbit 18, be randomly divided into 6 groups, respectively EXO freeze-dried powder group, EXO group, hASC freeze-dried powder group,
HASC group, physiological saline group, BSA positive controls (5mgBSA), rabbit two sides quadriceps muscle of thigh drug administration by injection 1ml, one time a day, even
3 days continuous, last 1 administration visually observes injection site and neighbouring muscle situation afterwards for 24 hours, then clip musculature is (from injection point
1cm) it is pathological section HE.Muscular irritation reaction grade scale: 0 grade, no significant change；1 grade, mild hyperaemia, range is 0.5
× 1.0cm or less；2 grades, moderate is congested, and range is in 0.5 × 1.0cm or more；3 grades, severe hyperemia is with myodegeneration；4 grades,
There is necrosis, there is brown denaturation；5 grades, there is popularity necrosis.
1.2.6.3 hemolysis in vitro is tested
After 2kg male rabbit 1, raising 3 days, arteria carotis communis is taken blood (about 20ml), continuous with the wooden stick for being tied with absorbent cotton
Blood is stirred, fibrinogen is removed, makes defibrinated blood.About 10 times of physiological saline amounts are added, shake up, centrifugation (1500
Turn 15min), supernatant is removed, the red blood cell of precipitating is washed 2 times according to the above method with physiological saline, until the not aobvious red of supernatant
Until.Gained red blood cell is made into the suspension that volume fraction is 2% with physiological saline, is divided into every pipe 2.5ml.EXO is lyophilized
Powder group, EXO group, hASC freeze-dried powder group, hASC group, physiological saline group, distillation water component are not added in test tube, 37 DEG C of incubations,
Haemolysis and red blood cell condensation phenomenon are visually observed, is centrifuged (1500 turns of 15min) after 3h, counting red corpuscles are resuspended in cell precipitation
Number, takes supernatant to measure light absorption value under 545nm.
1.2.6.4 Active general anaphylaxis
Male SD rat 36 (200g) are taken, be randomly divided into 12 groups: excretion body freeze-dried powder high dose group, excretion body are lyophilized
Powder low dose group, excretion body high dose group, excretion body low dose group, hASC high dose group, hASC low dose group, hASC freeze-drying
Powder high dose group, hASC freeze-dried powder low dose group, physiological saline group, BSA positive controls (1mg), BSA positive controls
(5mg), BSA positive controls (2.5mg).
Each group tested material and Freund's complete adjuvant are mixed in equal volume as suspension.Sensitization: the next day be injected intraperitoneally each group suspension
1ml, totally 3 times.Excitation: 14 days after injecting for the first time, every group took 1 rat, and for intravenous injection each group by test solution 1ml, observation half is small
When interior cavy performance.Performance :-asymptomatic；+ uneasiness trembles, scratches nose, sneeze, urinates, being short of breath；++ remove above-mentioned disease
Outside shape, expiratory dyspnea and dyskinesia；+++ in addition to above-mentioned symptom, respiratory failure can restore up to spasm；++++dead.
1.2.6.5 the passive hypersensitive test of skin
Male SD rat 12 (200g) are taken, is randomly divided into 12 groups (being grouped with upper section), each group tested material and Freund are complete
Adjuvant is mixed in equal volume as suspension.Sensitization: the next day be injected intraperitoneally each group suspension 1ml, totally 3 times.It is adopted after 14 days after sensitization for the first time
Blood prepares antiserum.Male SD rat 12 are taken, each group antiserum stoste is intradermal in back part of animal backbone two sides hair removal section
Inject 0.1mL/ point, after 2 days, groups of animals carry out antigen attack: tail vein injection 1ml (0.5ml antigen+0.5ml10% she
Text this blue solution) challenging antigen, animal is put to death after 30 points, extract skin of back, taken pictures and to measure the blue on the inside of skin anti-
Answer spot diameter.
Embodiment
Embodiment 1. freezes the morphologic observation of hASC and hASC freeze-dried powder
As a result as shown in Figure 1, in which: figure A, B are the cellular morphology that the hASC of rehydration is lyophilized；Scheming C, D is freeze-drying water again
Cellular morphology after the hASC culture for 24 hours of change；Scheme cellular morphology when E, F are conventional cryopreservation hASC recovery；It is conventional for scheming G, H
Cellular morphology after freezing hASC culture for 24 hours is in shuttle shape compact growth.
As can be seen that commonly freeze human adipose-derived stem cell recovery when, cell is full, rounded, 37 DEG C culture for 24 hours
It is afterwards in shuttle shape adherent growth.Compared with commonly freezing hASC, the hASC form that rehydration is lyophilized is more irregular, negligible amounts, portion
Cell is divided to become fragment.For 24 hours, freeze-drying rehydration hASC is not adherent for culture, loses cell activity.
The BCA protein content that embodiment 2. freezes EXO and EXO freeze-dried powder compares
As a result as shown in Figure 2, in which: figure A in 5 groups of samples be respectively labeled as sample (sample) -1, sample-2,
Sample-3, sample-4, sample-5, the initial excretion body equivalent of every group of sample；Figure B is the excretion body freeze-dried powder that BCA is measured
From the excretion body frozen stock solution protein content that (20-25 DEG C) different storage times are surveyed at room temperature.
As schemed shown in A, the 562nm OD value with EXO freeze-dried powder in group slightly above freezes EXO, i.e., albumen measured amount compared with
It is high.Be thought of as excretion body has part EXO bimolecular film broken in freeze-drying process, leads to wherein albumen spilling, albumen is caused to measure
Value increases.Figure B shows that the protein content of excretion body freeze-dried powder extends reduction less at any time, and excretion body frozen stock solution albumen contains
Amount changes over time sharp fall, is thought of as caused by the cracking of excretion body protein.
The measurement of the size distribution of embodiment 3.hASC excretion body and freeze-dried powder
As a result as shown in Figure 3, in which: A, B, C are the NTA data of excretion body；D, E, F are excretion body freeze-dried powder rehydration
NTA data.
The diameter of excretion body focuses mostly in 130nm or so, and the partial size of excretion body freeze-dried powder focuses mostly on 150nm or so.EXO
The partial size of freeze-dried powder is slightly larger compared with EXO, and partial elastic may be lost during freeze-drying by being thought of as EXO bimolecular film, leads to film
Relaxation, partial size become larger.
The test of the vascular stimulation of embodiment 4.hASC excretion body and freeze-dried powder
As a result are as follows: EXO freeze-dried powder group, EXO group, hASC freeze-dried powder group, hASC group and negative control physiological saline group are administered
Without significant reaction, evaluation with the naked eye is 0 grade at position.From pathological examination as can be seen that EXO freeze-dried powder group, EXO group, hASC freeze
Dry powder group, hASC group, physiological saline group endothelium are complete, without hyperemia, denaturation, necrosis etc., illustrate EXO freeze-dried powder, EXO, hASC
The equal vascular irritation of freeze-dried powder, hASC.
The muscular irritation of embodiment 5.hASC excretion body and freeze-dried powder is tested
As a result are as follows: EXO freeze-dried powder group, EXO group, hASC freeze-dried powder group, hASC group and negative control physiological saline group endothelium
It is complete, without hyperemia, denaturation, necrosis etc., it is chosen as 0 grade, BSA group has moderate congested, is chosen as 2 grades.Illustrate EXO freeze-dried powder,
EXO, hASC freeze-dried powder, hASC are without muscle irritation.
The test of the hemolysis in vitro of embodiment 6.hASC excretion body and freeze-dried powder
As a result as shown in Figure 4, in which: figure A be test result photo, each test tube be followed successively by EXO freeze-dried powder group, EXO group,
HASC freeze-dried powder group, hASC group, physiological saline group, distilled water group；Figure B be centrifuged after each group supernatant 545nm OD value, every group
3 samples；Scheming C is red blood cell count(RBC) after being resuspended.
In A figure, preceding 5 pipe supernatant is limpid, and cell precipitation is more；The 6th red dye of pipe supernatant, precipitating is less, illustrates distilled water group
There is obvious haemolysis, other each group haemolysis are unobvious (Fig. 4-A).Each group takes supernatant to survey 545nmOD value, distillation after centrifugation
Water positive group OD value is higher, there is obvious haemolysis, other group of OD value is lower, and haemolysis is unobvious (Fig. 4-B).Microscope after resuspension
Lower carry out red blood cell count(RBC), distilled water positive group red blood cell is minimum, other group of red blood cell is obviously higher than distilled water group (Fig. 4-
C).Result above prompts, and EXO, EXO freeze-dried powder, hASC, hASC freeze-dried powder not will cause extensive haemolysis.
The Active general anaphylaxis of embodiment 7.hASC excretion body and freeze-dried powder
As a result as shown in Figure 5, in which: 1-12 is respectively excretion body freeze-dried powder high dose group, excretion body freeze-dried powder low dosage
Group, excretion body frozen stock solution high dose group, excretion body frozen stock solution low dose group, hASC high dose group, hASC low dose group, hASC
Freeze-dried powder high dose group, hASC freeze-dried powder low dose group, physiological saline group (negative control), BSA positive controls (1mg),
BSA positive controls (2.5mg), BSA positive controls (5mg), every group of 3 rats.
The results show that 1-8 group is negative, 10-12 positive controls are positive.EXO, EXO freeze-dried powder, hADC, hASC
Freeze-dried powder Active general anaphylaxis is negative, and does not cause Ι type hypersensitivity, and safety is reliable.
The passive cutaneous anaphylaxis test of embodiment 8.hASC excretion body and freeze-dried powder
As a result as shown in Figures 6 and 7, in which: 1-12 is respectively that excretion body freeze-dried powder high dose group, excretion body freeze-dried powder are low
Dosage group, excretion body frozen stock solution high dose group, excretion body frozen stock solution low dose group, hASC high dose group, hASC low dose group,
HASC freeze-dried powder high dose group, hASC freeze-dried powder low dose group, physiological saline group (negative control), BSA positive controls
(1mg), BSA positive controls (2.5mg), BSA positive controls (5mg), ruler it is measured for locus coeruleus maximum in skin most
Major diameter；Data in Fig. 7 are derived from data measured by the ruler of Fig. 6, to be surveyed locus coeruleus maximum gauge.
The results show that 1-8 group skin locus coeruleus maximum gauge is respectively less than 10-12 positive controls diameter, illustrate EXO, EXO
Freeze-dried powder, hADC, hASC freeze-dried powder Active general anaphylaxis are negative, and will not cause obvious dermoreaction.
The rehydration process of comparative example .hASC excretion body freeze-dried powder
The present inventor further investigated the concentration of rehydration solution and in the freeze-drying method of cell it is common
Various other rehydration solutions, such as 5% sucrose, 5% skimmed milk power, 5% cellobiose and 5%D- galactolipin are obtained to final
The influence of the hASC excretion body freeze-dried powder arrived.It was found that the effect of the hASC excretion body freeze-dried powder obtained using other rehydration solutions
Fruit is obviously not so good as the hydroxyethyl starch solution that the present invention uses；And the concentration of hydroxyethyl starch solution of the invention is also required to
Smaller range, such as 5.6-6.3%, preferably 5.9-6.2% are maintained, most preferably 6.0% can obtain optimal effect.
It discusses
The present inventor has studied the effect of fat stem cell first.Even if it was found that in trehalose and human serum albumin
Protection under, after the freeze-dried rehydration process of fat stem cell, show that cellular portions are broken under microscope, but still have part thin
Born of the same parents have eucaryotic cell structure, but form size is irregular, and cell space owes plentiful.After conventional overnight incubation, rehydration cell is lyophilized
Without near-wall air curtain.Illustrate under this condition, though cellular morphology can be kept, being lost after the freeze-dried rehydration of fat stem cell
Cell activity.Therefore the freeze drying process is unsuitable for the storage of fat stem cell.
Rich in substances such as a large amount of protein and RNA in excretion body surface face and vesica, and played by these albumen and RNA
Biological effect, laboratory can pass through the content of the content detection excretion body of protein.Therefore, although the jelly of fat stem cell
Dry research is without obtaining expected success, but the present inventor does not abandon, and then studies the excretion body in fat stem cell source
Effect.The BCA Protein Detection of excretion body and excretion body freeze-dried powder is the results show that the freeze-dried guarantor of equivalent amount excretion body
It deposits higher than conventional cryopreservation protein content measured value.The egg that the surveyed excretion body protein content of BCA reagent is exposed by excretion body surface face
It is white.After freeze-drying saves the higher freeze-dried rehydration processing suspected of excretion body of albumen measured value, partial rupture causes egg in vesica
White spilling increases BCA measured value.Partial size is slightly larger after the freeze-dried rehydration of excretion body as the result is shown by NTA, but diameter still exists
Within 50-200nm.It is influenced suspected of excretion body by freeze-drying rehydration, phospholipid bilayer film original structure and loss of elasticity cause
Excretion volume morphing is irregular, and front volume is relatively lyophilized and increases, but vesica is still complete unbroken.Result above prompts just short-term protect
For depositing, freeze drying technology is slightly inferior to conventional cryopreservation technology, it is contemplated that freeze-drying operation itself is more more complicated than freezing operation, to equipment
Requirement it is higher, corresponding cost is also higher, it appears that by freeze drying technology be applied to excretion body storage prospect it is bad.
It is explored however, the present inventor increases the holding time to being lyophilized and freezing two methods.Respectively at 0,2,4,6,
8,10,7 time points of December carry out BCA determining the protein quantity, discovery freeze-drying skill to excretion body freeze-dried powder and excretion body frozen stock solution
Art storage excretion body protein measured amount it is constant, and conventional cryopreservation storage excretion body since 2 months albumen measured amount gradually under
Drop.In general, excretion body freeze drying technology is more suitable for long-term preservation.
Since the substances such as trehalose, hydroxyethyl starch are added in freeze-drying rehydration process of the invention, and excretion body is freeze-dried
Partial crushing, form also change after rehydration, so the safety after excretion body is freeze-dried needs to assess.This research
Vascular stimulation tests, flesh have been carried out respectively to excretion body freeze-dried powder, excretion body, fat stem cell freeze-dried powder, fat stem cell
Meat irritation test, hemolytic test, the test of active systemic anaphylaxis, passive cutaneous anaphylaxis test.The results show that excretion
Body freeze-dried powder, excretion body, fat stem cell freeze-dried powder, fat stem cell do not cause a positive reaction.Fat stem cell and excretion
Body immunogenicity is low, is not easy to cause immunological rejection, after freeze-dried, fat stem cell and excretion body freeze-dried powder it is immune
Originality finds no raising phenomenon.So it could be assumed that, excretion body freeze-dried powder, excretion body, fat stem cell freeze-dried powder, rouge
Fat stem cell without blood vessel, muscle irritation, will not cause haemolysis, can be used for body injection, Active general anaphylaxis, quilt
Dynamic skin anaphylactic test feminine gender explanation does not cause Ι type hypersensitivity, and immunogenicity is low, and safety is reliable.
Excretion body can be caused to cause excretion body portion broken though this research institute obtains excretion body freeze-dried powder, form also becomes
Change, but still the integrality of a large amount of excretion bodies can be kept, can long-term preservation, securely and reliably.Illustrate that excretion body freeze drying technology has into one
Step explores the value utilized, or can reach better effect by changing freeze-drying pretreatment condition.
All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet
It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Claims (10)
1. a kind of lyophilized preparation of the excretion body in human stem cell source, the lyophilized preparation includes the excretion body in human stem cell source
And trehalose.
2. lyophilized preparation as described in claim 1, which is characterized in that the lyophilized preparation is obtained by the following method:
1) solution of the solution of excretion body and trehalose is mixed to get to the mixed solution of the two；
2) the resulting mixed solution of step 1) is lyophilized, to obtain lyophilized preparation described in claim 1.
3. lyophilized preparation as claimed in claim 1 or 2, which is characterized in that surveyed compared to the original excretion body protein before storage
It must measure, the lyophilized preparation at room temperature, such as stores 4 months, 5 months, 6 months, 7 months, 8 months, 9 at 25-25 DEG C
Month, 10 months, 11 months or after 12 months, excretion body protein measured amount is higher than 95%, 96%, 97%, 98%, even higher than
99%.
4. a kind of pharmaceutical composition, described pharmaceutical composition includes lyophilized preparation of any of claims 1-3 and appoints
The pharmaceutically acceptable excipient of choosing.
5. a kind of rehydration object of the lyophilized preparation of the excretion body in human stem cell source, which is characterized in that the human stem cell comes
The lyophilized preparation of the excretion body in source is lyophilized preparation of any of claims 1-3.
6. a kind of treat with kit, the kit is equipped with:
1) lyophilized preparation of any of claims 1-3 or pharmaceutical composition as claimed in claim 4；
2) 5.6-6.3%, preferably 5.9-6.2%, most preferably 6.0% hydroxyethyl starch solution；With
3) using the hydroxyethyl starch solution described in 2) make 1) described in lyophilized preparation or pharmaceutical composition rehydration and will
To rehydration object give this needs object operation instructions.
7. a kind of preparation method of the lyophilized preparation of the excretion body in human stem cell source of any of claims 1-3,
It the described method comprises the following steps:
1) solution of the solution of excretion body and trehalose is mixed to get to the mixed solution of the two；
2) the resulting mixed solution of step 1) is lyophilized, to obtain the lyophilized preparation.
8. a kind of preparation method of the rehydration object of the lyophilized preparation of the excretion body in human stem cell source described in claim 5,
It is characterized in that, with 5.6-6.3%, preferably 5.9-6.2%, most preferably 6.0% hydroxyethyl starch solution, preferably at 37 DEG C
The lyophilized preparation of the excretion body in rehydration human stem cell source of any of claims 1-3, so that preparation is described again
Hyrate.
9. described in the lyophilized preparation or claim 5 of the excretion body in human stem cell source of any of claims 1-3
Human stem cell source excretion body lyophilized preparation rehydration object purposes in medicine preparation.
10. purposes as claimed in claim 9, which is characterized in that the drug is treatment acute hepatic failure, liver fibrosis, liver
Hardening, the preferably drug of acute hepatic failure.
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