CN107022648A - A kind of cellular resources store-service method - Google Patents
A kind of cellular resources store-service method Download PDFInfo
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Abstract
The present invention provides a kind of cellular resources store-service for the public, the healthy cell resource of itself or allosome is stored, it can be taken out when itself or relatives are ill for treating, for a insurance of life storage, the various information of personally identifiable information and storage cell is associated, user is directly inquired about by long-range cloud server and obtains information, provide a user conveniently systematization cellular resources store-service, user only need to provide cell derived tissue samples and relevant information in service point, so that it may which cellular resources depth is long-term stored in cell bank.
Description
Technical field
The invention belongs to cellular resources store-service technical field, specifically, it is related to a kind of cellular resources store-service
Method.
Background technology
Recently as the development of modern medicine, the appearance of cell therapy technology so that medical technology, which is constantly obtained, to be rouse oneself
The progress of the popular feeling.Cell therapy technology is broadly divided into stem-cell therapy and the major class of immunocyte two.
Cellular replacement therapy refers to, in healthy stem cell transplantation to patient's body, repair or replace impaired thin to reach
Born of the same parents or tissue, so as to reach the purpose of healing.Because stem cell can be divided into a variety of functioning cells or histoorgan, stem cell moves
Plant therapeutic domain very wide, can typically treat the nervous system disease, disease of immune system, also have some other Medicine and Surgery diseases.
Immune cell therapy technology effectively can remove and kill cancer cell by adjusting immune system.Be referred to as after traditional operation, radiotherapy,
The 4th kind of tumor therapeuticing method after chemotherapy.
Cell storage refers to the cell of health is carried out into deep hypothermia preservation, dimension by certain technology by certain method
Holding the function and activity of cell is not influenceed significantly.The healthy cell storage of itself or allosome is got up, can be at itself
Or cell therapeutic approach is enabled during relatives' illness, some illnesss is effectively treated, be a insurance of life storage.
As cell therapy technology is increasingly mature, cell storage expense declines to a great extent and expanded demand, the cell money of the public
Source store-service demand is increasingly apparent, then needs to set up the cellular resources store-service method of set of system, fan out from point to area clothes
The public in regional extent of being engaged in, meets the demand of cellular resources store-service.
The content of the invention
Instant invention overcomes shortcoming of the prior art there is provided a kind of cellular resources store-service method, by itself or
The healthy cell resource of allosome is stored, and can very easily be taken out when itself or relatives are ill for treating, is life
A insurance of storage.
In order to solve the above-mentioned technical problem, the present invention is achieved by the following technical solutions：
A kind of cellular resources store-service method, it is characterised in that comprise the following steps：
1) cellular resources store-service point is set up；
2) client provides cell derived tissue samples and blood sample to be checked in above-mentioned service point, and gathers client's personal identification letter
Breath；
3) cell derived tissue samples transport cell and prepare center, and cell prepares and is centrally generated sample cell unique identifier
And it is associated with personally identifiable information；Detection to cell derived tissue samples and blood sample to be checked；Cell derived tissue samples
Handle and therefrom separate target cell；The expansion culture of target cell；The Characteristics Detection of target cell；Target cell freezes；
Target cell is stored into cell bank profound hypothermia；
4) the year detection of target cell quality；
5) every terms of information and data upload cloud server；
6) client according to unique identifier beyond the clouds server obtain data result.
Further, the cellular resources include adult stem cell, iPS cells or immunocyte, the adult stem cell bag
Include mescenchymal stem cell, candidate stem cell or NSC.
Further, step 1) in, the cellular resources store-service point is arranged on hospital, school, beauty parlor, community, with
Principle nearby makes the public obtain service.
Further, the step 2) described in cell derived tissue samples include umbilical cord, placenta, fat, amniotic fluid, dental pulp,
Skin, uterine decidua, bleeding of the umbilicus, peripheral blood or menses.
Further, step 3) during the cell derived tissue samples transport cell preparation center, tissue samples are put
It is placed in special sample transport device, transport whole process is monitored equipped with humiture tape deck to tissue samples, it is ensured that group
Knit the biological activity of sample.
Further, step 3) in, the detection project of blood sample to be checked includes blood routine detection, herpesviral (EB)；Giant cell
Viral (CMV)；AIDS virus (HIV)；Microspironema pallidum (TP)；Hepatitis B (HBV)；Hepatitis C virus (HCV)；Thermophilic T cell disease
The detection of malicious (HTLV).
Further, step 3) in, separation target cell method have enzyme digestion, tissue block adherent method or density gradient from
Heart method, described enzyme digestion is to utilize digestive ferment tissue samples, and tissue samples are dissociated into individual cells, described digestive ferment
For trypsase, clostridiopetidase A or hyaluronidase, described tissue block adherent method is to shred tissue samples for 1-2mm3Size
Tissue block, be then directly inoculated in Tissue Culture Flask or culture plate cultivate, cell can be swum out of along tissue block edge, described
Density-gradient centrifugation method is used for the processing of blood sample and therefrom obtains target cell, refers to blood sample being added in inertia gradient
Centrifugal sedimentation or sedimentation equilibrium are carried out in medium, different cells are assigned to some certain bits in gradient under certain centrifugal force
Put, form the separation method of different zone.
Further, step 3) in, the step of expansion of target cell is cultivated is：Primary subject cell fusion rate reaches
80%, attached cell is departed from into blake bottle or culture dish bottom using digestive ferment, supernatant is removed after centrifugation, and add newly
Cell is resuspended in cell culture medium, is inoculated in new Tissue Culture Flask or is passed on, and carries out amplification cultivation；Hereafter per 2-5 days
Liquid is changed once until after fusion rate reaches 80%, producing, being passed on again if necessary；
The Characteristics Detection project of target cell：Cell quantity is determined, cellular activity is determined, cell-specific surface markers
Detection, Bacteria Detection, fungal detection, detection of mycoplasma or endotoxin detection；
The cryopreservation step of target cell is prepares deepfreeze under the conditions of tissue freezing solution, 4 DEG C, then by often pipe 1mL points
It is attached in cryopreservation tube, gradient cooling is carried out to -80 DEG C using program cooling system；
Described cell bank is described into soma to preserve Various Tissues derived adult stem cells, iPS cells or immunocyte
Cell includes：Umbilical cord, placenta, fat, amniotic fluid, dental pulp, skin, uterine decidua, bleeding of the umbilicus, peripheral blood or menses, cell bank are provided with
Temperature stabilization maintains -196 DEG C in liquid nitrogen container, liquid nitrogen container, can ensure that substantially change does not occur for the function and activity of cell for a long time
Change.
Further, step 4) in the annual detection project of cell quality to include cell survival rate, cell recoveries, growth bent
Line, culture form or specific cell surface markers, in addition stem cell also need to detect its differentiation capability or Colony forming ability.
Further, step 5) described in every terms of information include personally identifiable information, target cell Characteristics Detection result, mesh
Mark the testing result and the particular location of cell storage of the detection of cell quality year and cell derived tissue samples and blood sample to be checked
And cell storage state.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention provides a kind of cellular resources store-service for the public, and the healthy cell resource of itself or allosome is stored
Come, can be taken out when itself or relatives are ill for treating, be a insurance of life storage, by personally identifiable information and storage
The various information of cell is associated, and user is directly inquired about by long-range cloud server and obtains information, provides a user conveniently
Efficiently systematization cellular resources store-service, user only need to provide cell derived tissue samples and relevant information in service point,
Cellular resources depth can be long-term stored in cell bank.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, together with embodiments of the present invention for explaining the present invention,
It is not construed as limiting the invention, in the accompanying drawings：
Fig. 1 is a kind of flow chart of cellular resources store-service of the present invention.
Embodiment
The preferred embodiments of the present invention are illustrated below in conjunction with accompanying drawing, it will be appreciated that preferred reality described herein
Apply example to be merely to illustrate and explain the present invention, be not intended to limit the present invention.
As shown in figure 1, a kind of cellular resources store-service of the present invention comprises the following steps：
1) cellular resources store-service point is set up
Cellular resources store-service point is generally located on hospital, school, beauty parlor, community etc., makes public affairs with principle nearby
Crowd obtains service.
Wherein, the cellular resources store-service point has multiple, and multiple cellular resources store-service points are to put into
The mode in face is distributed in the region of effective transportation radius, and drives the public in region on the cellular resources store-service point
Obtain service.
2) client provides cell derived tissue samples and blood sample to be checked in cellular resources store-service point
Client provides the target cell derived tissues sample and blood sample to be checked to be stored in cellular resources store-service point.
Cell derived tissue samples can be umbilical cords, placenta, fat, amniotic fluid, dental pulp, skin, uterine decidua, bleeding of the umbilicus, outer
All blood, menses equal samples.
Blood sample to be checked is extracts each 2 pipes of client's peripheric venous blood 5ml, and a pipe contains anti-coagulants, another to contain anti-coagulants, respectively
Washed corpuscles and serum, the detection for follow-up indices.
Gather client's personally identifiable information
Staff's collection of cellular resources store-service point and typing client's personally identifiable information, the identity information bag
Include：Name, contact method, the data such as heredity medication history.
3) cell derived tissue samples transport cell and prepare center
During cell derived tissue samples transport cell preparation center, tissue samples are positioned over special sample transport
In device, transport whole process is monitored equipped with humiture tape deck to tissue samples, it is ensured that the biological activity of tissue samples.
Generate sample cell unique identifier and associated with personally identifiable information
Sample cell unique identification can be Quick Response Code, bar code, can also make other character marks, but these are identified
Should be associated with the personally identifiable information of the client.
The detection of cell derived tissue samples and blood sample to be checked
The detection project of tissue samples includes Sterility testing and fungal detection.The detection project of blood sample to be checked includes blood routine
Detection, herpesviral (EB)；Cytomegalovirus (CMV)；AIDS virus (HIV)；Microspironema pallidum (TP)；Hepatitis B (HBV)；
Hepatitis C virus (HCV)；The detection of thermophilic T cell viral (HTLV).Above items detection can pass through the qualified mechanism of third party
Detection.
The processing of umbilical cord sample and therefrom separating mesenchymal stem cell
Umbilical cord tissue sample is as a kind of cell derived tissue samples, a kind of target cell of storage of mescenchymal stem cell.
Total length about 5cm umbilical cord tissue sample, surface is taken to cut into the small of appropriate length after being cleaned through 75% ethanol disinfection
Blood vessel in section, the amnion and umbilical cord on separation umbilical cord surface, which is torn, to be taken the jelly of Wharton tissue in umbilical cord and cleans, and uniformly shreds into 1-
2mm3Fritter.Balance weighs 3g jelly of Wharton tissue pieces, is separately added into 10ml complete mediums.Training completely is added before absorption
The people's umbilical cord jelly of Wharton tissue kind for supporting base enters Tissue Culture Flask.
The processing of placental samples and therefrom separating mesenchymal stem cell
Placenta tissue sample is as a kind of cell derived tissue samples, a kind of target cell of storage of mescenchymal stem cell.
Operating theater instruments completely separates the fine hair membrane tissue of placenta tissue edge connection and is attached to tissue above, uses tissue
Protect liquid cleaning blood and blood clot.It is with operating theater instruments scraping decidua vera tissue, fine hair membrane tissue and amnion tissue, its is clear
Wash.After cleaning, 3g tissue mass is collected respectively in centrifuge tube.Tissue is shredded, NTx enzyme, 37 DEG C 1500rpm/ points is added
Clock concussion digestion 2h.Terminated and digested with 5ml complete mediums, tissue fluid after digestion is diluted to 100ml with organization protection's liquid.Will
Centrifuge tube is put into centrifuge, parameter of noncentricity：2000rpm,10min.Supernatant is abandoned in shifting, is diluted to again with organization protection's liquid
100ml.Cell suspension is centrifuged again, parameter of noncentricity：1200rpm,10min.Supernatant is abandoned in shifting, is resuspended with 10ml complete mediums
Cell precipitation obtains cell suspension.The cell suspension of chorion, decidua vera and amnion is planted into a cell bottle respectively.
The processing of amniotic fluid sample simultaneously therefrom separates amniotic fluid stem cell
Amniotic fluid sample is as a kind of cell derived tissue samples, a kind of target cell of storage of amniotic fluid stem cell.
Aseptically, first 10ml amniotic fluid sample is filtered by using 200 mesh standard sieves, it is broken with the tissue for removing larger
Piece.1000r/min, centrifuges 10min, and abandoning supernatant collects lower confluent monolayer cells；Amniotic fluid sample protection liquor is added, mixes, puts
37 DEG C of processing 5min；1000r/min, centrifuges 10min, and abandoning supernatant collects lower confluent monolayer cells；Add amniotic fluid sample protection liquid molten
Liquid, is mixed, and puts 37 DEG C of processing 5min；1000r/min, centrifuges 10min, and abandoning supernatant collects lower confluent monolayer cells, adds amniotic fluid and do
It is inoculated in after cell special culture solution in cell bottle.
The processing of tooth samples simultaneously therefrom separates dental pulp stem cell
Tooth samples are as a kind of cell derived tissue samples, a kind of target cell of storage of dental pulp stem cell.
Children's deciduous teeth or adult impacted wisdom tooth are taken, is cut away corona with gear drill is vertical or horizontal in culture dish, with taking
Broach removes pulp tissue.The pulp tissue of taking-up is put into centrifuge tube.With physiological saline rinse culture dish 2-3 times, collect
Rinse liquid, is transferred into Ep pipes.Pulp tissue is suitably shredded with eye scissors, with digestive juice 1:1 mixing, 37 DEG C of digestion 1h.Gently
Tissue block is blown and beaten, makes cell dissociation into individual cells suspension.Brine 1-2 times, 2000rpm, 10min remove supernatant.With
Containing complete medium be resuspended cell, be inoculated in blake bottle or in.
The processing of fatty sample and therefrom fractionation of fatty stem cell
Fatty sample is as a kind of cell derived tissue samples, a kind of target cell of storage of fat stem cell.
30g adipose tissues are placed in collecting bottle 50ml physiological saline, are acutely rocked 3min fully to wash adipose tissue, are connect
Static 3-5min, makes different phase separations, lower floor's aqueous phase is sucked；Operation 3-5 times more than repeating, until subnatant is more limpid.
Pipette, removes suction nozzle, and first drawing lower floor's red liquid in fat acquisition bottle is discarded, and remaining upper-layer fat is dispensed after mixing.
Adipose tissue mixture is dispensed into two 50ml centrifuge tubes.Add isometric collagenase solution, 37 DEG C, 150rpm, digestion
Until seeming more smooth.Isolation medium vascular component (SVF)：2000rpm centrifuges 10min.It is small from top to bottom with pipette
The heart removes upper strata grease and the collagenase solution of lower floor.Cell is resuspended in physiological saline, dispels, and postdigestive tissue is used into 100um
After cell strainer filtering, 300g centrifugations 10min.Stuffing fat stem cell media is fully mixed after centrifugation, is inoculated in cell culture
In bottle.
The processing of peripheral blood sample and therefrom isolating immune cells
Peripheral blood sample is as a kind of cell derived tissue samples, a kind of target cell of storage of immunocyte stem cell.
20ml human lymphocyte separating liquids are added in centrifuge tube, separation liquid pipe are made, normal temperature is standby.The blood that PBS suspends is thin
Born of the same parents are slowly added to lymphocyte separation medium, and 2000rpm centrifugations 20min (rises 7 drops 0)；Divide from top to bottom in centrifuge tube after centrifugation
For four layers：First layer is plasma layer；The second layer is ring-type milky buffy coat；Third layer is separation liquid layer；4th layer is
Red blood cell layer；Second layer tunica albuginea confluent monolayer cells are collected, the 50ml centrifuge tubes to 2 cell washing solutions containing 5-10ml are respectively added slowly to
In, soft piping and druming is mixed, and cell is resuspended with cell washing solution and mends to 50ml, 1800rpm centrifugations 10min；Abandon supernatant, Xiang Qi
In plus 10ml blanc cell nutrient solution X-VIVO15, cell suspension is made, mix.
The expansion culture of mescenchymal stem cell
Mesenchymal cell stem cell is used as a kind of target cell of storage.
Primary mescenchymal stem cell cell confluency reaches 80% or so, departs from attached cell using digestive ferment and cultivates
Bottle or culture dish bottom, remove supernatant after centrifugation, and add new cell culture medium resuspension cell, are inoculated in new cell training
Support bottle or passed on, and carry out amplification cultivation；Hereafter per changing within 2-5 days after liquid once reaches 80% up to fusion rate, produce, must
Passed on again when wanting.
Expand cultural method be equally applicable to amniotic fluid stem cell, dental pulp stem cell, fat stem cell, skin progenitor cell or its
The expansion culture of his attached cell.
The Characteristics Detection of mescenchymal stem cell
The Characteristics Detection project of target cell：Cell quantity is determined, cellular activity is determined, cell-specific surface markers
Detection, Bacteria Detection, fungal detection, detection of mycoplasma, endotoxin detection etc..
Cell quantity is carried out using cell counter and determines what is determined with cellular activity, using flow cytomery cell
Specific surface marker, Bacteria Detection, fungal detection, detection of mycoplasma, endotoxin detection can be sent to the qualified machine of third party
Structure is detected.
Target cell freezes
The cryopreservation step of target cell is prepares deepfreeze under the conditions of tissue freezing solution, 4 DEG C, then by often pipe 1mL points
It is attached in cryopreservation tube, gradient cooling is carried out to -80 DEG C using program cooling system.
It is resuspended, adjustment cell density is 2 × 106/mL, is distributed into cryopreservation tube, is covered, and do using cells frozen storing liquid
Good numbering, carries out program mode cooling using programmed cooling instrument immediately,；
Program is as follows：Prior to 4 DEG C balance 20min, are then down to -5 DEG C, then with 21 DEG C/min with 1 DEG C/min speed
Speed be down to -50 DEG C, then be warming up to -21 DEG C with 10 DEG C/min speed, be then down to -40 DEG C with 2 DEG C/min speed, connect
And be down to -80 DEG C with 10 DEG C/min speed.
Target cell is stored into cell bank profound hypothermia
- 80 DEG C in embodiment 15 of cell is placed in -196 DEG C of cell banks in liquid nitrogen container, depth storage records cell
The position of storage.
4) the year detection of target cell quality
The annual detection project of cell quality includes cell survival rate, cell recoveries, growth curve, culture form, spy
Specific cell-surface markers etc., in addition stem cell also need to detect its differentiation capability or Colony forming ability.
Cell survival rate and cell recoveries are carried out using cell counter, using flow cytomery specific cell
Surface markers, growth curve determines the cck-8 methods that use, culture form by micro- sem observation, stem cell differentiation capability by into
Fat, skeletonization and into cartilage three be differentiation judge.
5) every terms of information and data upload cloud server
Every terms of information include personally identifiable information, target cell Characteristics Detection result, target cell quality year detection and
Particular location and cell storage state of the testing result and cell storage of cell derived tissue samples and blood sample to be checked etc..
According to unique identifier, server obtains data result to client beyond the clouds
6) client obtains the every terms of information and number of registration by sample cell unique identifier on long-range cloud server
According to result.
Finally it should be noted that：The preferred embodiments of the present invention are these are only, are not intended to limit the invention, although
The present invention is described in detail with reference to embodiment, for those skilled in the art, it still can be to foregoing
Technical scheme described in each embodiment is modified, or to which part technical characteristic carry out equivalent substitution, but it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc. should be included in the protection of the present invention
Within the scope of.
Claims (10)
1. a kind of cellular resources store-service method, it is characterised in that comprise the following steps：
1) cellular resources store-service point is set up；
2) client provides cell derived tissue samples and blood sample to be checked in above-mentioned service point, and gathers client's personally identifiable information；
3) cell derived tissue samples transport cell prepare center, cell prepare be centrally generated sample cell unique identifier and with
Personally identifiable information is associated；Detection to cell derived tissue samples and blood sample to be checked；The processing of cell derived tissue samples
And therefrom separate target cell；The expansion culture of target cell；The Characteristics Detection of target cell；Target cell freezes；Target
Cell is stored into cell bank profound hypothermia；
4) the year detection of target cell quality；
5) every terms of information and data upload cloud server；
6) client according to unique identifier beyond the clouds server obtain data result.
2. a kind of cellular resources store-service method according to claim 1, it is characterised in that the cellular resources include into
Somatic stem cell, iPS cells or immunocyte, the adult stem cell include mescenchymal stem cell, candidate stem cell or nerve cord
Cell.
3. a kind of cellular resources store-service method according to claim 1, it is characterised in that step 1) in, the cell
Resource store-service point is arranged on hospital, school, beauty parlor, community, the public is obtained service with principle nearby.
4. a kind of cellular resources store-service method according to claim 1, it is characterised in that the step 2) described in it is thin
Born of the same parents' derived tissues sample includes umbilical cord, placenta, fat, amniotic fluid, dental pulp, skin, uterine decidua, bleeding of the umbilicus, peripheral blood or menses.
5. a kind of cellular resources store-service method according to claim 1, it is characterised in that step 3) cell derived
During tissue samples transport cell preparation center, tissue samples are positioned in special sample transport device, and transport is whole
Tissue samples are monitored equipped with humiture tape deck, it is ensured that the biological activity of tissue samples.
6. a kind of cellular resources store-service method according to claim 1, it is characterised in that step 3) in, blood sample to be checked
Detection project include blood routine detection, herpesviral (EB)；Cytomegalovirus (CMV)；AIDS virus (HIV)；Treponemal
Body (TP)；Hepatitis B (HBV)；Hepatitis C virus (HCV)；The detection of thermophilic T cell viral (HTLV).
7. a kind of cellular resources store-service method according to claim 1, it is characterised in that step 3) in, separate target
The method of cell has enzyme digestion, tissue block adherent method or density-gradient centrifugation method, and described enzyme digestion is to utilize digestive ferment
Tissue samples are dissociated into individual cells by tissue samples, and described digestive ferment is trypsase, clostridiopetidase A or hyaluronidase,
Described tissue block adherent method is to shred tissue samples for 1-2mm3The tissue block of size, is then directly inoculated in cell culture
Cultivated in bottle or culture plate, cell can be swum out of along tissue block edge, described density-gradient centrifugation method is used for the place of blood sample
Manage and therefrom obtain target cell, refer to blood sample being added in progress centrifugal sedimentation or sedimentation equilibrium in inertia gradient media,
Different cells are assigned in gradient on some ad-hoc locations under certain centrifugal force, the separation method of different zone is formed.
8. a kind of cellular resources store-service method according to claim 1, it is characterised in that step 3) in, target cell
Expansion be the step of cultivate：Primary subject cell fusion rate reaches 80%, departs from attached cell using digestive ferment and cultivates
Bottle or culture dish bottom, remove supernatant after centrifugation, and add new cell culture medium resuspension cell, are inoculated in new cell training
Support bottle or passed on, and carry out amplification cultivation；Hereafter per changing within 2-5 days after liquid once reaches 80% up to fusion rate, produce, must
Passed on again when wanting；
The Characteristics Detection project of target cell：Cell quantity is determined, cellular activity is determined, the inspection of cell-specific surface markers
Survey, Bacteria Detection, fungal detection, detection of mycoplasma or endotoxin detection；
Then the cryopreservation step of target cell is dispensed into prepare deepfreeze under the conditions of tissue freezing solution, 4 DEG C by every pipe 1mL
In cryopreservation tube, gradient cooling is carried out to -80 DEG C using program cooling system；
Described cell bank is preservation Various Tissues derived adult stem cells, iPS cells or immunocyte, the adult stem cell
Including：Umbilical cord, placenta, fat, amniotic fluid, dental pulp, skin, uterine decidua, bleeding of the umbilicus, peripheral blood or menses, cell bank are provided with liquid nitrogen
Temperature stabilization maintains -196 DEG C in tank, liquid nitrogen container, can ensure that significant change does not occur for the function and activity of cell for a long time.
9. a kind of cellular resources store-service method according to claim 1, it is characterised in that step 4) in cell quality
Annual detection project includes cell survival rate, cell recoveries, growth curve, culture form or specific cell surface markers,
Other stem cell also needs to detect its differentiation capability or Colony forming ability.
10. a kind of cellular resources store-service method according to claim 1, it is characterised in that step 5) described in it is each
Item information includes personally identifiable information, target cell Characteristics Detection result, the detection of target cell quality year and cell derived group
Knit sample and the testing result of blood sample to be checked and the particular location of cell storage and cell storage state.
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