CN103469309A - Method for separating living cell and constructing cell bank by means of tissue homogenate method - Google Patents
Method for separating living cell and constructing cell bank by means of tissue homogenate method Download PDFInfo
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Abstract
The invention discloses a method for separating the tissue cell of a fetal appendage and constructing a cell bank by means of a tissue homogenate method, so that the process for constructing the cell blank is simple in steps, time-saving, and capable of achieving easy quality control and standardization. The method disclosed by the invention comprises the following steps of (1) cleaning the tissue of the fetal appendage; (2) carrying out homogenate pretreatment on the tissue; (3) carrying out homogenate treatment on the tissue to separate a cell (cell cluster); (4) filtering the tissue and collecting the cell (cell cluster) after the homogenate; (5) detecting the cell (cell cluster); (6) cryopreserving the separated cell (cell cluster) and constructing the cell bank. Compared with the method in the prior art, the method disclosed by the invention has the advantages of simple operation, short time consumption, no introduction of an exogenous reagent, easiness for standardization, easy quality control, great cell gain and the like, and is of great significance in improving the construction efficiency of the fetal appendage cell bank and obtaining the stem cell which is high in quality and applied to treatment.
Description
Technical field
The present invention relates to the construction process in fetal appendage histocyte storehouse, more specifically say a kind of physical method from fetal appendage separate tissue cell construction cell (group) storehouse.
Background technology
Fetal appendage is organized mainly and is consisted of placental lobules, placenta base, basal decidua, amnion and umbilical cord tissue.The placenta that originates from the embryonic development period extraembryonic mesoderm is comprised of interstitial, blood vessel and nurse cell, is the hemocytopoietic organ of fetus very early time, contains a large amount of interstitial compositions.Up-to-date research shows in placenta to contain the inferior myeloid-lymphoid stem cell of mescenchymal stem cell, placenta and abundant hemopoietic stem cell, and from placenta, separation and Culture goes out these multipotential stem cells and will open up a brand-new and abundant source for experimental study and clinical application.
The inferior myeloid-lymphoid stem cell of placenta derives from placenta tissue, from embryoplastic the 5th to 7 days, starts to occur, can be differentiated to form 200 organ cells of various human body tissue, but can not form a complete human body.The many biological properties that possess multipotential stem cell, this cells in vitro can be to directed differentiation such as adipocyte, scleroblast, neurocyte, liver epithelial like cells.
The placenta Hematopoietic Stem is thin
born of the same parents arebeing present in a group primitive hematopoietic cell in placenta tissue, is the first ancestor of hemocyte (red corpuscle, white corpuscle, thrombocyte etc.), is the height undifferentiated cell, also can say that it is the initiating cell of all hemocytes (wherein great majority are immunocytes).In placenta tissue the content of hemopoietic stem cell be hemopoietic stem cell content in Cord blood 5-10 doubly, can be personal several times for child, or offer the treatment of 1-2 adult patient.Efficiently solve marrow or the rear derived from peripheral blood deficiency of mobilization while transplanting, Cord blood quantity waits technical barrier not simultaneously, will be expected to replace marrow, mobilize rear peripheral blood and Cord blood for hematopoietic stem cell transplantation.
When basal decidua is mother's gestation, the trophocyte of blastocyst, once contacting with uterine endometrium, causes that near the inner membrance mesenchymal cell hypertrophy it is bred the tissue formed.Basal decidua forms placental septa chorion frondosum is separated into to several fine hair leaflets, wherein contains the basal decidua stem cell in mother source.
Amnion (amnion) is the thin film that covers the amniotic cavity internal surface, from cytotrophoblast, develop, it is the internal layer of the two-layer fetal membrane of the mankind, its thickness is 0.02～0.05mm, it is basilar membrane the thickest in human body, by amniotic epithelial cells (human amniotic epithelial cell, HAEC), basilar membrane, essential layer, fibroblast layer, 5 layers of structure of spongy layer, formed.People's amniotic epithelial cells is positioned at the innermost layer of amnion, amniotic epithelial cells is grown from epiblast at after fertilization on the 8th day, make it may maintain the plasticity-that gastrula forms the pre-embryo stem cell, have unique using value aspect treatment nervus centralis obstacle: 1996, Sakuragawa etc. found people's amniotic epithelial cells expression neurone of cultivation and the sign of neurogliocyte at first.Research subsequently shows, people's amniotic epithelial cells of cultivation can synthesize and release neurotransmitters as vagusstoff, catecholamine and Dopamine HCL.The experiment of people's amniotic epithelial cells transplantation treatment Parkinson disease in rats (PD) shows that it can not only survive and express TYR hydroxylase (TH) activity in vivo, and can reverse disease and prevent neuronal death.People's amniotic epithelial cells has good therapeutic action equally for the Spinal injury of primates.
Umbilical cord is the tie that fetus is connected with parent.In umbilical cord, have three blood vessels to pass through, a radicular vein and two radicular arterieses, play the sanguimotor effect between mothers and sons of linking up.The umbilical cord of term fetus is about 30～70 centimetres.Stem cell in umbilical cord China Tong Shi glue (Wharton ' s Jelly) is mainly umbilical cord mesenchymal stem cells.
Mescenchymal stem cell (mesenchymal stem cell, MSC) is a kind of multipotential stem cell, derives from mesoderm in fetal development.In the normal tissue injury repair process of body, MSC is a kind of cell of important participation tissue regeneration.Under the distinctive signal effect caused in tissue injury, MSC moves to damaged part, assembles propagation in part, according to different damage signals, along different approaches, breaks up.MSC is easy to separate amplification, and external multiplication ability is vigorous, even increase 100,000,000 times, still can keep its Multidirectional Differentiation ability.Therefore, MSC is a kind of tissue repair seed cell of practicality.
MSC has powerful multiplication capacity and multi-lineage potential, in suitable body or under external environment, has and is divided into: epithelial stem cell, neural stem cell, liver stem cells, the ability of the various kinds of cell such as myocyte, scleroblast, chondrocyte, stroma cell.Can be used for repairing the histoorgan of impaired or pathology, the various diseases such as the treatment heart, cerebrovascular disease, nervous system disorders, hepatic diseases, osseous tissue disease, corneal injury, empyrosis, myopathy; And, MSC has immunoregulation effect, by the negativity immunoloregulation function, suppress the hyperfunction immune response of body, body's immunity is restored balance, thereby can be used for treating immunological rejection after hematopoietic stem cell transplantation and clone disease, lupus erythematosus, the autoimmunization systemic diseases such as scleroderma; The important component part of MSC or human body microenvironment, transplant mescenchymal stem cell and can change hematopoieticmicroenviron-ment, rebuilds immunity system, promotes hematopoietic function recovery, with the hemopoietic stem cell co-transplantation, can significantly improve the result for the treatment of of leukemia and refractory anemia etc.
The structure of fetal appendage cell bank; by former neonatal umbilical cord that Biohazard Waste directly disposes and the placenta tissue of being used as by processing; extract wherein abundant stem cell resource; and make it to store for a long time under dormant state by ripe deep-frozen technology; Biological resources deposit as a kind of preciousness; in the future, can be applied to the treatment of many autologous major diseases; Simultaneously, also can be for transplanting and the treatment of allosome; Can also be developed to stem cell drugs by researching and developing again and processing, benefit the whole society.
The fetal appendage cell bank builds the separate tissue that comprises cell, purifying, freezing preservation, the aspects such as the detection of pathogenic micro-organism and quality control.The related fetal appendage cell bank construction process of this patent is the technology that in wherein organizing, method, the especially cell of cellular segregation separates from solid tissue.From tissue, the method for isolated cell structure cell bank has enzyme solution and cell mass culture method at present.Wherein the enzyme solution is the main method widely adopted.
The enzyme solution is the method for utilizing digestive ferment that active somatic cell is separated from tissue.About 1-1.5mm first is cut into tissue shear in concrete sepn process
3fritter, then adopt collagenase or collagenase and pancreatin to be used in combination to carry out cell mass digestion, and utilize the enzymic digestion stop buffer such as serum to stop digestion, finally filter to clean and obtain cell.The patent of grant number CN101608174B " a kind of construction process of human umbilical cord mesenchyma stem cell " for example, the separation of mescenchymal stem cell is exactly the method that adopts collagenase digesting.The patent of grant number CN100344757C " people's placenta, Umbilical Filling stem cell's storehouse library And Construct method ", build method that the mescenchymal stem cell in storehouse adopts and be collagenase and pancreatin digestion method successively.The enzyme solution has following characteristics: 1), because needs are used a large amount of enzymes and digestion stop buffer, experimental cost is higher; 2), due to different working method and personnel's situation, each cell yield obtained differs greatly.3) due to the digestion method difference adopted, the time of whole digestion enzymolysis is different in size, from 30 minutes, by several hours, has.
The cell mass culture method is a kind of physics and the method that cultivation combines cell is separated from tissue, is also to build a kind of available method of cell bank.About 1-1.5mm first is cut into tissue shear in concrete sepn process
3fritter, be inoculated into culture of isolated cell in culture dish.The patent of grant number CN102010850B " a kind of umbilical cord tissue mescenchymal stem cell creep plate separate method " for example, the separation of umbilical cord tissue mescenchymal stem cell is exactly the cell mass culture method adopted.The method has following characteristics: 1) only need tissue shear is cut into to 1-1.5mm
3cell mass and simple the cultivation, operate fairly simple; 2) cultivation of the cell mass needs time of several days could be realized the separation of cell, consuming time longer; 3) long-term cultivation of cell, the possibility of pollution is larger.
As can be seen here, the solid tissue cell isolation method operating process that builds at present cell bank is more loaded down with trivial details, length consuming time, and the manual operation that places one's entire reliance upon, high to technician's operating skill requirement.In addition, the enzyme solution has been introduced again the exogenous agents of animal-origin, can add the risk of the stem cell drugs treatment of big bus cell bank.
Guarantee that in the present invention is based on the cell bank structure cell isolation method is simple, save time, be easy to Quality Control and normalized principle, adopt tissue homogenate method isolating fetal appurtenant tissue, obtain a large amount of for building the cell in storehouse, to meet scientific research, medicine and the field such as the clinical demand to the cell bank resource.
Summary of the invention
The purpose of this invention is to provide a kind of tissue homogenate isolating fetal appurtenant cell (group) and set up the method for cell bank.
The histocyte processor P lacentaPro that the present invention preferably uses is a kind of instrument that the invention people designs for the present invention, this instrument comprises control device and homogenate container two portions, as shown in Figure 1, control device comprises fuselage (1), homogenate container putting hole (2), rotating head (3), draw-in groove (4), louvre (5); The homogenate container comprises cup (6), bowl cover (7), sealing-ring (8), rotating head junctor (9), stationary shaft (10), cutter head (11), dop (12).
Fuselage (1) side is provided with louvre (5), fuselage (1) top is homogenate container putting hole (2), homogenate container putting hole (2) inwall is provided with draw-in groove (4), homogenate container putting hole (2) bottom is rotating head (3), when processing is organized, the homogenate container is placed in homogenate container putting hole (2), rotating head junctor (9) lower end of bowl cover (7) bottom engages with rotating head (3), upper end is connected with cutter head (11) by stationary shaft (10), bowl cover (7) is by sealing-ring (8) and cup body (6) tight joint, the dop (12) of cup body (6) outer side wall engages with draw-in groove (4).
The cutter head (11) that wherein key part is particular design.Cutter head (11) is four leaf blades, by stacked on top of one another, is placed and mutually perpendicular blade (13) and blade (14) form.Blade (13) divides 5 sections, comprises 4 bendings, and the bending of two, two ends is 130-150 degree (preferably 138 degree), and middle two bendings are 140-160 degree (preferably 148 degree); Blade (14) divides 3 sections, comprises 2 bendings, and two bendings are 110-130 degree (preferably 123 degree);
During work, by the tissue block glass body (6) of packing into, after covering bowl cover (7), the homogenate container is put into to homogenate container putting hole (2), moving by some biographies of controller-rotating head (3)-rotating head junctor (9)-stationary shaft (10)-cutter head (11).Band dynamic cutter head (11) rotates, thereby plays the homogenate effect.
Although a small amount of histocyte processor P lacentaPro effect similar instrument used to the present invention arranged in the market, as the gentleMACS sample disposal device of Germany U.S. sky Ni company also can chorista obtain cell suspension.But the first, the treatment capacity of existing instrument less all, generally can only process the following sample of 5 grams on the market, and the multipotency of PlacentaPro is processed the sample of 400 grams, for setting up cell bank and mass cell, cultivates and has established good basis; Second, existing instrument is due to structural limitations on the market, can only process breakable tissue, as spleen, liver, lung etc., for thering is the very fetal appendage tissue of obdurability, as the umbilical cord that contains a large amount of magnificent Tong Shi glue is also helpless, and PlacentaPro is due to the singularity of structure, can be in the situation that do not destroy cytoactive, the large tissue by toughness, as the fetal appendage tissue carries out homogenate, directly separate and obtain cell/cell mass.
Technical scheme of the present invention comprises the process that cleaning, homogenate pre-treatment, tissue homogenate processing, filtered and recycled and the cell bank of tissue build, and concrete steps are as follows:
(1) tissue cleans: by appropriate scavenging solution (for example, containing two anti-PBS damping fluids), the fetal appendage tissue obtained is rinsed, until the remained blood of tissue surface is rinsed totally.
(2) tissue homogenate pre-treatment: according to the size of the homogenate container of histocyte processor P lacentaPro; the tissue obtained is cut into small pieces; put into the histocyte treater, add damping fluid (for example PBS damping fluid) the seal tissue cellular processor of 0.5-2 times of quality;
(3) tissue homogenate is processed: the rotation that histocyte processor P lacentaPro drives special cutter head by rotor will be organized chopping and homogenate, and tissue is carried out to cellular segregation and extraction;
(4) tissue filter is processed, and cell harvesting: the fragment of tissue that step (3) is obtained is transferred to filtration unit, and placenta tissue is ground, and collects filtrate, and filtrate is carried out to cell cleaning, centrifugal collecting cell; To the cell mass on the umbilical cord tissue de-entrainment filter, clean cell mass and collect.
(5) cell (group) separated builds cell bank: the cell (group) that step (4) is separated is put liquid nitrogen and is preserved, and by its ABO/Rh blood group and HLA somatotype, is preserved, and foundation can, for the cell news file of retrieval, be constructed cell bank.
The described need of the step according to the present invention (1) sample to be processed is the fetal appendage tissue, comprises placental lobules and basal decidua tissue, placenta base tissue, placenta amnion tissue and umbilical cord tissue.
The tissue sample size of the described processing of the step according to the present invention (2) is:
Placental lobules and basal decidua 30-120g, preferred 60-90g, the damping fluid added or the volume of substratum are 20-110ml, preferably 50-80ml;
Umbilical cord 5-30g, preferred 10-20g, the damping fluid added or the volume of substratum are 10-45ml, preferably 15-30ml;
Placenta base 100-300g, preferred 150-200g, the damping fluid added or the volume of substratum are 100-300m1, preferably 150-200ml;
Placenta amnion 5-25g, preferred 10-20g, the damping fluid added or the volume of substratum are 10-40ml, preferably 15-25ml;
The described histocyte processor P of the step according to the present invention (2) lacentaPro processes:
Placental lobules and basal decidua organized processing rotating speed are 1000-2400rpm, preferably, and 1400-1800rpm;
It is 1500-3500rpm that umbilical cord tissue is processed rotating speed, preferably, and 2500-2800rpm;
Placenta base organized processing rotating speed is 1500-2500rpm, preferably, and 2000rpm;
Placenta amnion organized processing rotating speed is 2000-3000rpm, preferably, and 2300-2500rpm;
The described fragment of tissue filtration unit of the step according to the present invention (4) is 100-400 order metallic filter, preferred 200 purpose strainers when placental lobules and basal decidua tissue, placenta base tissue filter, preferred 400 purpose strainers when umbilical cord tissue, placenta amnion tissue filter.
Beneficial effect of the present invention is: the method that the present invention adopts the histocyte processor P lacentaPro of independent development to be separated the fetal appendage histocyte, have simple to operate, the used time is short, without exogenous agent, add, be easy to the advantages such as stdn and quality control and cell acquisition amount are large, for improving the structure efficiency of cell bank, ensure that the quality of cell bank high-quality has great importance.
The accompanying drawing explanation
Fig. 1 histocyte processor P lacentaPro structural representation
Fig. 2 primary cell culture form
Fig. 3 umbilical cord tissue is processed the cell mass obtained and is cultivated and reach the P3 cell cultures aspect graph in generation
Fig. 4 umbilical cord tissue is processed the cell mass obtained and is cultivated and reach the P3 cell streaming figure in generation
The former culture aspect graph of the cell mass obtained after Fig. 5 placenta amnion organized processing
Embodiment
Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: adopt the tissue homogenate method to separate placental lobules cell construction cell bank
The method of placental lobules tissue homogenate comprises the following steps:
(1) placenta tissue cleans: placenta tissue is processed in Biohazard Safety Equipment, use in right amount and containing 1% pair of anti-PBS damping fluid, placenta tissue is rinsed according to the placenta size, to placenta tissue remained on surface blood, rinse well, the placenta surface is without blood clotting;
(2) the placental lobules tissue is processed early stage: the placenta tissue that uses operating scissors to obtain from step (1) is cut placental lobules; placental lobules is transferred on culture dish and is cut into small pieces; the placental lobules tissue block of 60-90 gram is put into to the homogenate container of histocyte processor P lacentaPro, added PBS the encapsulation process cup of 50-80 milliliter;
(3) the placental lobules tissue homogenate is processed: the placental lobules tissue block of utilizing histocyte processor P lacentaPro that step (2) is obtained is shredded by tissue and cellular segregation and extraction are carried out in the physical treatment of homogenate, stirring velocity is between 1400rpm-1800rpm, and the treatment time is 5 minutes;
(4) the placental lobules tissue filter is processed: the placental lobules tissue after step (3) is disposed is transferred on 200 order metallic filters, fragment of tissue is ground and utilizes another culture dish to collect the liquid filtered, be divided into 2 times and add the PBS cleansing tissue of 10-15ml and continue to grind toward metallic filter.
(5) cell counting: collect with the 50ml centrifuge tube cell suspension that step (4) obtains, with the speed of 1400rpm/ minute centrifugal 10 minutes, remove supernatant and add the PBS re-suspended cell, extract sample in a small amount and carry out cell counting.
Placental lobules histocyte separating treatment test is divided into 8 groups to be carried out respectively, and each group has been carried out two different testing setups, is respectively (a) 1400rpm, 5 minutes and (b) 1800rpm, 5 minutes.Placenta tissue derives from 8 different donors, and treatment capacity is between 60 gram left and right.Experimental result is as shown in table 1.
Table 1 (a) placental lobules tissue homogenate is processed the cell extraction data
Table 1 (b) placental lobules tissue homogenate is processed the cell extraction data
As can be seen from the above results, full-automatic placenta tissue device is (a) 1400rpm in test condition, 5 minutes is (b) 1800rpm with test condition, processes the placental lobules tissue under the setting of 5 minutes, and the every gram karyocyte mean number finally obtained is respectively 2.98 * 10
6with 2.8 * 10
6, the cell quantity that both obtain does not have significant difference, and the lifting that has proved speed does not have clear and definite linear relationship to the efficiency of cell extraction.And, in supplementary test, by fluidic cell, test discovery, the cell of cell surface marker CD34 positive expression wherein is respectively 2.92 * 10
4/ g and 2.78 * 10
4/ g; Another supplements in experiment, too high and too low rotating speed and homogenate time improper all can shadow to the efficiency of cell extraction.Test and can find by survival rate, separate and the karyocyte that obtains still keeps good vigor through the placenta tissue treater, 8 test result survival rates can reach more than 98%.Above result shows, full-automatic histocyte processor P lacentaPro can effectively separate and extract karyocyte from the placental lobules tissue, and simple, consuming time short, for the structure of cell bank provides effective guarantee.
Embodiment 2: the comparative experiments of the processing of placental lobules tissue homogenate and manual process
Comparative experiments divides 8 groups to carry out.The method of reference example 1 is carried out full-automatic organized processing experiment.Method described in referenced patent CN201210292509.9 (publication number CN102807966A) is carried out manual process (digestion) experiment.Placenta tissue is derived from 8 different donors, and treatment capacity is between the 60-90 gram.Contrast and experiment is as shown in table 2.
| ? | Full automatic treatment | Manual process (digestion) |
| Test | Extract cell count/g | Extract cell count/ |
| 1 | 3.8×10 6／g | 2.0×10
6／ |
| 2 | 3.9×10 6／g | 0.79×10
6／ |
| 3 | 2.3×10 6／g | 1.0×10
6／ |
| 4 | 2.9×10 6／g | 1.1×10
6／ |
| 5 | 2.6×10 6／g | 1.4×10
6／ |
| 6 | 2.7×10 6／g | 1.1×10
6／ |
| 7 | 3.1×10 6／g | 0.86×10
6／ |
| 8 | 2.5×10 6／g | 1.7×10 6／g |
| On average | 2.98×10 6／g | 1.24×10 6／g |
Table 2 full automatic treatment and manual process placental lobules organising data contrast table
Result shows, every gram average cell amount that the placental lobules tissue obtains after full-automatic histocyte processor P lacentaPro processes is 2.4 times of manual process, process the gained cell apparently higher than modified manual digestion, for the foundation of cell bank provides a large amount of cells, saved the time of follow-up cell amplification.
Embodiment 3: the primary growing state of cell that the placental lobules tissue separates through the homogenate method
(1) before cell cultures, prepare: with 1 part of cell suspension, than the ratio of 2-3 part erythrocyte cracked liquid, add erythrocyte cracked liquid (Roche), cultivate 10-15 minute under the environment of 15-25 ℃, put whizzer into the speed of 1400rpm centrifugal 10 minutes, remove supernatant, observe the erythrocyte splitting situation, repeat again this step if needed and carry out cracking.After cracking, add the PBS re-suspended cell and extract sample in a small amount and carry out cell counting, put into whizzer with centrifugal 10 minutes of the speed of 1400rpm to clean cell, remove supernatant, add mescenchymal stem cell substratum (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) re-suspended cell, and with 2-5 * 10
4/ cm
2density placenta source karyocyte is inoculated in culturing bottle.
(2) primary cell culture: culturing bottle is put into to C0
2concentration is 5%, and the incubator that temperature is 37 ℃ is cultivated, and partly changes for the first time liquid while being cultured to the 6th day, partly changed for the second time liquid at the 9th day, at the 12nd day, the substratum of plate the inside is taken away, again added the 15ml substratum, within every 2-3 days backward, once entirely change liquid.Cultivate form by microscope observing cell, the cell attachment growth is typical fusiformis, and it is the inoblast sample that the endochylema projection is arranged, and cytoplasm is abundant, and kernel is obvious, is the growth of whirlpool shape.(seeing Fig. 2)
(3) passage: the attached cell fusion rate when the culturing bottle the inside reaches 80% left and right, can utilize digestive ferment (TrypLE Express) that attached cell is broken away to the culturing bottle bottom, centrifugally supernatant is taken away and added cell special culture media re-suspended cell afterwards, being inoculated in Tissue Culture Flask is gone down to posterity and is continued amplification cultivation, within every 2 days backward, change liquid once until after fusion rate reaches 80%, obtain, gone down to posterity again in case of necessity.
8 groups of cells that extract for embodiment 3 carry out former culture, and growth result is as shown in table 3.
The primary growing state of cell that table 3 placental lobules separates
8 groups of placental lobuless separate the karyocyte that obtains in same culture conditions through histocyte processor P lacentaPro, and each primary growth conditions of organizing cell is basically identical, all at 12-14 days left and right primary cells, covers with.This consistence shows, full-automatic homogenate method is when carrying out extensive sample preparation, and the sample room otherness is little, and standardization level is high, has higher quality-guarantee.
Embodiment 4: adopt the tissue homogenate method to separate the umbilical cord cell mass
Umbilical cord tissue homogenate method comprises the following steps:
(1) umbilical cord tissue cleans: umbilical cord tissue is processed in Biohazard Safety Equipment; umbilical cord tissue is cut open; take out the blood vessel in umbilical cord; then with containing 1% pair of anti-PBS damping fluid, umbilical cord tissue being rinsed; to umbilical cord tissue remained on surface blood, rinse well, the umbilical cord surface is without blood clotting;
(2) umbilical cord tissue is processed in earlier stage: the umbilical cord tissue that step (1) is obtained is transferred on culture dish, use operating scissors that umbilical cord tissue is cut into to the 2cm*2cm fritter, the umbilical cord tissue piece of 10-20 gram is put into to the homogenate container of histocyte processor P lacentaPro, added the 20-30 milliliter containing 1% couple of anti-PBS and seal the homogenate container;
(3) umbilical cord tissue homogenized: the umbilical cord tissue that using-system cellular processor PlacentaPro obtains step (2) shreds by tissue and the cell mass separation and extraction is carried out in the physical treatment of homogenate, stirring velocity is 2500-2800rpm, and the treatment time is 10 minutes;
(4) umbilical cord tissue filtration treatment: the umbilical cord tissue after step (3) is disposed is transferred on 400 order metallic filters, and the cell mass on de-entrainment filter is divided into 2 physiological saline with 5ml and cleans cell mass.
(5) umbilical cord cell mass adherent culture primary cell: the cell mass that step (4) is obtained is layered in plate, add mescenchymal stem cell substratum (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12), plate is put into to CO
237 ℃ of incubators that concentration is 5% were cultivated, and partly change for the first time liquid while being cultured to the 6th day, partly changed for the second time liquid at the 9th day, at the 12nd day, started, and within every 2 to 3 days, once entirely changed liquid.Cultivate form by microscope observing cell, the cell attachment growth is typical fusiformis, and it is the inoblast sample that the endochylema projection is arranged, and cytoplasm is abundant, and kernel is obvious, is the growth of whirlpool shape.
(6) passage: the attached cell fusion rate when the plate the inside reaches 80% left and right, can utilize digestive ferment (TrypLE Express) that attached cell is broken away to the culturing bottle bottom, centrifugally supernatant is taken away and added cell special culture media re-suspended cell afterwards, being inoculated in Tissue Culture Flask is gone down to posterity and is continued amplification cultivation, within every 2 days backward, change liquid once until after fusion rate reaches 80%, obtain, gone down to posterity again in case of necessity.Fig. 3 is that the P3 cell cultures aspect graph in generation is cultivated and reached to the cell mass that the umbilical cord tissue processing obtains, and Fig. 4 is that the P3 cell streaming figure in generation is cultivated and reached to the cell mass that the umbilical cord tissue processing obtains.
Embodiment 5: adopt the tissue homogenate method to separate placenta base cell
The method of placenta base tissue homogenate comprises the following steps:
(1) placenta tissue cleans: placenta tissue is processed in Biohazard Safety Equipment, according to the placenta size, uses appropriate PBS damping fluid to be rinsed 2-3 time to placenta tissue, and placenta tissue remained on surface blood is rinsed well, and the placenta surface is without blood clotting;
(2) placenta base tissue is processed early stage: the placenta tissue that uses operating scissors to obtain from step (1) is cut the placenta base, the placenta base is transferred on culture dish and is cut into small pieces, placenta base tissue block between the 150-200 gram is put into to the homogenate container of histocyte processor P lacentaPro, added the PBS of 150-200 milliliter and seal the homogenate container;
(3) placenta base tissue homogenate is processed: the placenta base tissue that utilizes histocyte processor P lacentaPro that step (2) is obtained shreds by tissue and cellular segregation and extraction are carried out in the physical treatment of homogenate, stirring velocity is between 2000rpm, and the treatment time is 5 minutes;
(4) placenta base tissue filter is processed: the placenta base tissue after step (3) is disposed is transferred on 200 order metallic filters, fragment of tissue is ground and utilizes another culture dish to collect the liquid filtered, be divided into 2 times and add the PBS cleansing tissue of 20-25ml and continue to grind toward metallic filter.
(5) cell counting: collect with the 50ml centrifuge tube cell suspension that step (4) obtains, with the speed of 1400rpm/ minute centrifugal 10 minutes, remove supernatant and add the PBS re-suspended cell, extract sample in a small amount and carry out cell counting.
The test of placenta base histocyte separating treatment is divided into 8 groups to be carried out respectively, and test condition is 2000rpm, 5 minutes.Placenta tissue is derived from 8 different donors, and treatment capacity is between the 150-200 gram.Experimental result is as shown in table 4.
Table 4 placenta base tissue homogenate is processed the cell extraction data
As can be seen from the above results, histocyte processor P lacentaPro is 2000rpm in test condition, processes placenta base tissue under the setting of 5 minutes, and the every gram karyocyte mean number finally obtained is 6.1 * 10
5.In supplementary test, the improper efficiency that all can affect cell extraction of too high and too low rotating speed and homogenate time.Test and can find by survival rate, separate and the karyocyte that obtains still keeps good vigor through the histocyte treater, 8 test result survival rates can reach more than 99%.Above result shows, full-automatic histocyte processor P lacentaPro can effectively separate and extract karyocyte from placenta base tissue, and simple, consuming time short, for the structure of cell bank provides effective guarantee.
Embodiment 6: adopt the tissue homogenate method to separate the placenta amnion cell
Placenta amnion tissue homogenate method comprises the following steps:
(1) placenta tissue cleans: umbilical cord tissue is processed in Biohazard Safety Equipment, according to the umbilical cord size, uses appropriate PBS damping fluid to be rinsed 2-3 time to umbilical cord tissue, and umbilical cord tissue remained on surface blood is rinsed well, and the umbilical cord surface is without blood clotting;
(2) the placenta amnion tissue is processed early stage: the placenta tissue that uses operating scissors to obtain from step (1) is cut placenta amnion, amnion tissue is transferred on culture dish and is cut into small pieces, the placenta amnion tissue block of 10-20 gram is put into to the homogenate container of histocyte processor P lacentaPro, added the PBS of 15-25 milliliter and seal the homogenate container;
(3) the placenta amnion tissue homogenate is processed: the placenta amnion tissue that using-system cellular processor PlacentaPro obtains step (2) shreds by tissue and cellular segregation and extraction are carried out in the physical treatment of homogenate, stirring velocity is 2300-2500rpm, and the treatment time is 8 minutes;
(4) the placenta amnion tissue filter is processed: the placenta amnion tissue after step (3) is disposed is transferred on 400 order metallic filters, the cell mass on de-entrainment filter, minute 2 buffer solution for cleaning of the PBS with 5ml cell masses.
(5) primary cell culture: add amniotic epithelial cells substratum (10%FBS+90%DMEM+10ng/mlEGF+4mmo1/L glutamine) in the cell mass obtained in step (5), plate is put into to C0
2concentration was 5%, and the incubator that temperature is 37 ℃ is cultivated, and partly changes for the first time liquid while being cultured to the 6th day, partly changed for the second time liquid at the 9th day, at the 12nd day, started, and within every 2 to 3 days, once entirely changed liquid.Cultivate form by microscope observing cell, the cell attachment growth is full cobblestone-appearance, as shown in Figure 5.
Embodiment 7: fetal appendage tissue homogenate method isolated cell (group) frozen
Through the cell/cell mass of histocyte treater homogenized, filtration cleaning, if temporary transient inapplicable words can be carried out Cryopreservation, until after take out again the recovery cultivation while needing.
The cryopreservation methods of placenta cells is as follows:
Get 1 * 10
6cell joins in 1ml cells frozen storing liquid (containing 65 parts of DMEM-F12+10 part dimethyl sulfoxide (DMSO)+15 parts of human serum albumins), through programmed cooling, carries out freezing treatment, finally freezing sample is put into to liquid nitrogen container frozen.
The cryopreservation methods of umbilical cord cell mass is as follows:
Getting 1ml cells frozen storing liquid (containing 80 parts of human serum albumin+20 part dimethyl sulfoxide (DMSO)) adds in cryopreservation tube, the umbilical cord cell mass is added in frozen storing liquid, cumulative volume is no more than 1.8ml, through programmed cooling, carries out freezing treatment, finally freezing sample is put into to liquid nitrogen container frozen.
Embodiment 8: the foundation in fetal appendage cell (group) storehouse
1, the investigation of cell derived
Record placenta supplier (father and mother's) detail file, and place on record.
2, the detection of cell contamination
Utilize a small amount of cell cultures, detect the pollution whether cell is subject to fungus and bacterium.Utilize the etiology method, detect whether cell is subject to Hepatitis B virus, the third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST infects.
3, the detection of inherited disease
Utilize the method for molecular genetics, detect freeze-stored cell and whether have inherited disease.
4, HLA-ABC/DR joins type
Detect cell HLA-ABC/DR phenotype, and place on record.、
5, the detection of cytoactive
Utilize trypan blue staining to count the number of frozen front and back viable cell.
6, the foundation of placenta stem-cell database
After preserving normal placenta stem-cell, set up the database of placenta stem-cell, comprising the first five items data, and foundation and freeze-stored cell is associated.
Claims (8)
1. the method in a tissue homogenate method isolating fetal appurtenant tissue construction cell (group) storehouse is characterized in that being comprised of following step:
(1) to fetal appendage, organize the supplier to carry out ABO/Rh Blood grouping, the detection of HLA somatotype, microorganism immunodetection, with containing two anti-damping fluids, remove remained blood;
(2) tissue homogenate pre-treatment: the tissue after cleaning is cut into small pieces, puts into histocyte treatment unit sample chamber, add damping fluid or the substratum of 0.5-2 times of quality, the seal tissue cell handling device;
(3) tissue homogenate is processed: running histocyte treatment unit is 1000-3500rpm at rotating speed, under the condition that the treatment time is 1-15 minute, will organize chopping and homogenate, tissue is carried out to the separating treatment of cell (group);
(4) tissue filter is processed: the homogenate that step (3) is obtained is by 100-400 purpose strainer filtering: for placental lobules and basal decidua tissue, placenta base tissue, a little placenta tissue piece residual on filter screen is ground to collection filtrate; For umbilical cord tissue, placenta amnion tissue, collect the cell mass on filter screen.
(5) cell (group) is collected and is cleaned: for cell, with centrifuge tube, collect the cell suspension that step (4) obtains, with the centrifugal 5-15 minute of the speed of 1400-1800rpm/ minute, remove supernatant and add the damping fluid re-suspended cell; For cell mass, with direct collecting cell group after buffer solution for cleaning.
(6) cell (group) step (5) separated is put liquid nitrogen and is preserved, and by its ABO/Rh somatotype and HLA somatotype, is preserved, and foundation can, for the cell news file of retrieval, be constructed cell bank.
2. the method in tissue homogenate method isolating fetal appurtenant tissue construction cell as claimed in claim 1 (group) storehouse, it is characterized in that, in described step (1), the fetal appendage tissue comprises placental lobules and basal decidua, placenta base, placenta amnion and umbilical cord.
3. the method in tissue homogenate method isolating fetal appurtenant tissue construction cell as claimed in claim 1 (group) storehouse, it is characterized in that, in described step (2), the histocyte treatment unit used is the tissue homogenate treater, preferably the histocyte processor P lacentaPro of contriver's development.
4. the method in tissue homogenate method isolating fetal appurtenant tissue construction cell as claimed in claim 1 (group) storehouse, it is characterized in that, in described step (2), the size of the handled fetal appendage tissue sample of histocyte treatment unit is 5-300g, wherein umbilical cord tissue is 5-30g, placental lobules and basal decidua are organized as 30-120g, and the placenta base is organized as 100-300g, and placenta amnion is organized as 5-25g.
5. the method in tissue homogenate method isolating fetal appurtenant tissue construction cell as claimed in claim 1 (group) storehouse, is characterized in that, in described step (3), and placental lobules and the preferred 1000-2400rpm of basal decidua organized processing rotating speed; Umbilical cord tissue is processed the preferred 1500-3500rpm of rotating speed; The preferred 1500-2500rpm of placenta base organized processing rotating speed; The preferred 2000-3000rpm of placenta amnion organized processing rotating speed.
6. the method in tissue homogenate method isolating fetal appurtenant tissue construction cell as claimed in claim 1 (group) storehouse, it is characterized in that, in described step (4), when filtering placental lobules and basal decidua tissue, placenta base and organizing, preferred 200 purpose metal screens; When filtering umbilical cord tissue, placenta amnion and organizing, preferred 400 purpose metal screens.
7. the method in tissue homogenate method isolating fetal appurtenant tissue construction cell as claimed in claim 1 (group) storehouse, it is characterized in that, in described step (6), cell bank can be umbilical cord mesenchyma stem cell, placenta hemopoietic stem cell bank, the inferior totipotent cell of placenta storehouse, basal decidua stem cell bank and amniotic epithelial cells storehouse.
8. the method in tissue homogenate method isolating fetal appurtenant tissue construction cell as claimed in claim 1 (group) storehouse, is characterized in that, in described step (6), quality control has comprised the investigation of cell derived; The detection of cell contamination, as Hepatitis B virus, the third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis; Inherited disease detects; HLA-ABC/DR joins type and detects; The detection of cytoactive.
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| HK14105416.9A HK1192290A1 (en) | 2013-09-12 | 2014-06-10 | Method for separating living cell and constructing cell bank by means of tissue homogenate method |
| TW103131629A TW201510223A (en) | 2013-09-12 | 2014-09-12 | Method for separating living cell and constructing cell bank by means of tissue homogenate method |
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| CN104480533A (en) * | 2014-12-29 | 2015-04-01 | 黑龙江天晴干细胞股份有限公司 | Placenta stem cell bank construction method and placenta tissue resuscitation method |
| CN106399237A (en) * | 2016-11-30 | 2017-02-15 | 广州赛莱拉干细胞科技股份有限公司 | Method for primary isolation of umbilical cord mesenchymal stem cells |
| CN106801032A (en) * | 2017-02-17 | 2017-06-06 | 庞然 | The construction method of people's amnioic epithelium stem cell bank |
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| CN104480533A (en) * | 2014-12-29 | 2015-04-01 | 黑龙江天晴干细胞股份有限公司 | Placenta stem cell bank construction method and placenta tissue resuscitation method |
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| CN107541485A (en) * | 2017-10-19 | 2018-01-05 | 四川农业大学 | A kind of original, primary, secondary follicle the separation method of goose |
| CN108795853A (en) * | 2018-05-28 | 2018-11-13 | 天津博雅秀岩生物技术有限公司 | Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell |
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| CN110859856A (en) * | 2019-10-21 | 2020-03-06 | 中冠赛尔生物科技(北京)有限公司 | Transplant for reducing GVHD morbidity |
| CN112506620A (en) * | 2020-12-28 | 2021-03-16 | 网络通信与安全紫金山实验室 | Docker container deployment-based ospf protocol cleaning recovery method, device, equipment and medium |
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| CN114958717A (en) * | 2022-04-06 | 2022-08-30 | 中山大学附属第一医院 | Method for quickly separating living cells from trace calcified blood vessels |
| CN114958717B (en) * | 2022-04-06 | 2023-03-10 | 中山大学附属第一医院 | Method for quickly separating living cells from trace calcified blood vessels |
Also Published As
| Publication number | Publication date |
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| TW201510223A (en) | 2015-03-16 |
| TWI515299B (en) | 2016-01-01 |
| HK1192290A1 (en) | 2014-08-15 |
| CN103469309B (en) | 2015-12-23 |
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