CN107254446A - A kind of method for separating and preparing of people's primary tumor cell - Google Patents
A kind of method for separating and preparing of people's primary tumor cell Download PDFInfo
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Abstract
The invention provides a kind of method for separating and preparing of people's primary tumor cell, incubation is divided into two stages, and use the culture medium of the hyclone containing various concentrations to be cultivated respectively, by the concentration for gradually stepping up hyclone in culture medium, so that the adaptability of primary cell gradually strengthens, vigor is gradually stepped up, cell propagation is very fast, yield is higher, while genetic stability is good；The dense element of addition Sodium Pyruvate, mandarin orange, Y 27632 and griseofulvin, can prevent bacterium and fungal infection, and can promote cell propagation, suppress cell differentiation in the medium；Plant source recombination human serum albumin is added in frozen stock solution, hyclone can be partly replaced, the consumption of hyclone is reduced, so as to reduce cost in the case where ensureing that effect is constant.
Description
Technical field
The invention belongs to primitive cell culture technical field, prepared by the separation of more particularly to a kind of people's primary tumor cell
Method.
Background technology
Cell culture is one of biology and the most frequently used means of medical research, can be divided into original cuiture and Secondary Culture
Two kinds.Original cuiture is also referred to as Initial culture, is the part that tissue or organ are directly obtained from organism, through Mechanical Method or each
Enzyme (conventional trypsase) and intercalating agent (conventional EDTA) processing are planted, is allowed to be dispersed into individual cells, appropriate culture is added
Base, is placed in suitable culture vessel, under sterile, proper temperature and certain condition, is allowed to the mistake survived, grow and bred
Journey.
Strictly speaking, original cuiture refers to from internal tissue inoculated and cultured of taking out to passage stage first time；But it is actual
On, the culture cell within the first generation to the tenth generation is generally referred to as primitive cell culture.Original cuiture process is typically lasting
1~4 week, be in active movement in this phase cell, it is seen that cell division, but not vigorous.Primary cultured cell and internal basic stitch
Similarity degree is very high on morphosis and functional activity.Because the cell of culture is just separated from biological tissue, therefore
Closer to the animation in organism.This method can be provided with for the growth of research biological cell, metabolism, breeding
The means of power.Also created conditions simultaneously to carry out Secondary Culture later.
Existing primary culture method, is by adding serum in the medium come for cells with nutrient mostly.But blood
Clear cost is higher, if using the culture medium containing same concentration serum in incubation always, can cause cost expenses mistake
It is high, large-scale culture can not be carried out；If reducing the concentration of serum, nutrient supply deficiency, influence cell may be caused again
Propagation.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of method for separating and preparing of people's primary tumor cell.
Concrete technical scheme of the present invention is as follows：
The invention provides a kind of method for separating and preparing of people's primary tumor cell, comprise the following steps：
S1：The separation of primary tumor cell：Tumor tissues are gathered, are cleaned up, slough and connective tissue is removed,
And cut into tissue pieces；
S2：First cultivation stage：The tissue pieces are inoculated in Tissue Culture Flask, containing for 1~5mL are added low dense
The RPMI-1640 culture mediums of hyclone are spent, are cultivated 2~3 weeks；
S3：Second cultivation stage：The RPMI-1640 culture mediums containing high concentration hyclone are changed, continue to cultivate to thin
Born of the same parents' degrees of fusion reaches 70~80%；
S4：Cell harvesting and freeze：Obtained cell is subjected to digestion harvest, frozen stock solution is added into the cell of harvest,
And frozen.
Incubation is divided into two stages by this method, by gradually stepping up the concentration of hyclone in culture medium, so that
The adaptability of primary cell gradually strengthens, vigor is gradually stepped up, and cell proliferation is very fast, yield is higher, while effectively controlling into
This.
Further, the specific method of the step S1 is as follows：
Tumor tissues are gathered, are transferred under aseptic conditions in culture dish, with the chlorine that 5~10mL mass fractions are 0.9%
Change sodium water solution to rinse, repeat 2~3 times, remove bloodstain, and cut off slough and connective tissue, be cut into 1mm3Tissue it is broken
Block.
Tissue mass cell culture is the important method of cell primary culture, has the advantages that step is few, cellular damage is small, and
And culture medium need not be frequently changed, it is easy to operate.
Further, the step S2 comprises the following steps：
S2.1：Tissue pieces are inoculated in T25 blake bottles, the RPMI- of 1~5mL hyclones containing 50mg/L is added
1640 culture mediums, tissue block are uniformly laid on blake bottle bottom, are placed in 37 DEG C of CO2 incubators and are cultivated；
S2.2：A full dose was carried out every 3~4 days and changes liquid, old culture medium is discarded, while adding the fresh cultured of equivalent
Base, is placed in CO2 incubators and continues to cultivate；
S2.3：After culture 7~10 days, blake bottle is jiggled, tissue block is come off；By old culture medium and the group come off
The membrane filtration that block shifts and uses 100 μm in the lump is knitted, filtrate is collected, is centrifuged under the conditions of 1500~2000rpm/min
5min, collects precipitation, adds new culture medium, continues to cultivate 1~2 week.
Further, the specific method of the step S3 is as follows：
After first cultivation stage culture 2~3 weeks, old culture medium is discarded, the RPMI- containing 100mg/L hyclones is used instead
1640 culture mediums, carry out a full dose in every 3~4 days and change liquid, cultivate untill when cell fusion degree reaches 70~80%.
Further, the step S4 comprises the following steps：
S4.1：When cell total amount reaches 1*107When, old culture medium is discarded, is cleaned 2~3 times, abandoned with phosphate buffer
Remove cleaning solution；
S4.2：1~5mL 0.125~0.25% trypsase-EDTA digestive juices are added into the blake bottle after washing,
Gently shaking makes digestive juice infiltrate blake bottle bottom surface, is stood in superclean bench after 1min, takes out blake bottle and gently pats,
It is placed in observing under inverted microscope, untill when cell rounding, most cells come off；
S4.3：The RPMI-1640 culture mediums containing high concentration hyclone described in 3~5ml are added into blake bottle, will
The suspension transfer of cell and tissue block, will be sticked to thin in blake bottle with phosphate buffer described in 5~10mL in blake bottle
Born of the same parents are eluted, and the residual cells of elution are mixed with the suspension, and 5min is centrifuged under conditions of 1500~2000rpm/min；
S4.4：Cell is resuspended in supernatant discarding after centrifugation, phosphate buffer described in 5~10mL of addition, gently blows and beats mixed
It is even, the cell suspension after resuspension is centrifuged into 5min, and supernatant discarding under conditions of 1500~2000rpm/min；
S4.5：1~2mL frozen stock solutions are added into the cell being collected into, is transferred in cryopreservation tube, is put into program temperature reduction box,
- 80 DEG C of refrigerator freezings are placed in, cryopreservation tube is transferred in liquid nitrogen and preserved by next day.
Further, it is characterised in that the RPMI-1640 culture mediums are also added with following composition：100~150mg/L
The dense element of the mandarin orange of Sodium Pyruvate and 1~2mg/L.
Pyruvic acid is the intermediate product of glycolysis, can free in and out cell, can be the energy needed for metabolic activity in cells
Amount and carbon source, additionally aid the light toxic action that reduction fluorescent material triggers；And Sodium Pyruvate is most common acetonate, use
Sodium Pyruvate is added in culture medium instead of pyruvic acid, can be improved stability, be reduced cost.
The dense element of mandarin orange is a kind of natural mixture extracted from orange peel, and the effect with extremely strong antibacterial can suppress thin
The growing multiplication of bacterium etc., and have no toxic side effect, the growth of cell will not be impacted, it will not also produce pathogenic microorganism
The raw resistance to the action of a drug.
Further, following composition is also added with the RPMI-1640 culture mediums：4~6mg/L Y-27632 and
2~4mg/L griseofulvin.
Y-27632 is the related FZ kinase inhibitors of Rho, can suppress breaking up into fat for cell.Griseofulvin
The mitosis of fungi can effectively be suppressed, be clinically usually used in the fungal infections such as treatment favus of the scalp, tinea of feet and hands, tinea pedis and manus and trigger
Disease, be added in culture medium, can effectively prevent fungal contamination.
Further, the frozen stock solution includes following composition：Volume parts ratio is 5~7:2~4:1 hyclone, plant
Material resource recombination human serum albumin and dimethyl sulfoxide (DMSO).
Current conventional cryopreservation liquid mainly uses two kinds of compositions of hyclone and dimethyl sulfoxide (DMSO), cell preciousness degree is higher,
Requirement to nutrition is higher, and the consumption of hyclone is also higher；Plant source recombination human serum albumin is based on plant production system
Make, using the teaching of the invention it is possible to provide the nutritional ingredient suitable with serum, can partly substitute hyclone, thus reduce the consumption of hyclone,
Reduce cost.
Further, the duration of the step S4.2 is no more than 5min.
Digestion time is long, and trypsase may produce injury to cell, causes cell yield reduction, vigor to decline,
Generation influence is used on follow-up.
Beneficial effects of the present invention are as follows：, will the invention provides a kind of method for separating and preparing of people's primary tumor cell
Incubation is divided into two stages, and uses the culture medium of the hyclone containing various concentrations to be cultivated respectively, by by
The concentration of hyclone in culture medium is gradually improved, so that the adaptability of primary cell gradually strengthens, vigor is gradually stepped up, cell
Propagation is very fast, yield is higher, while genetic stability is good；The dense element of Sodium Pyruvate, mandarin orange, Y-27632 are added in the medium
And griseofulvin, bacterium and fungal infection can be prevented, and cell propagation can be promoted, suppress cell differentiation；In frozen stock solution
Plant source recombination human serum albumin is added, hyclone can be partly replaced, the consumption of hyclone is reduced, so as to protect
Cost is reduced in the case that card effect is constant.
Embodiment
The preparation of main agents and culture medium
A.RPMI-1640 culture mediums：Specific composition is as follows：
B. phosphate buffer：
Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4With 0.24g KH2PO4, it is dissolved in 800mL distilled water, uses
The pH value of HCl regulation solution finally adds distilled water to be settled to 1L to 7.4.In 15lbf/in2 (1034 × 105Pa) high pressure
Lower steam sterilization (at least 20 minutes), is stored in room temperature or 4 DEG C of refrigerators.
C. trypsase-EDTA digestive juices：
Weigh 0.125~0.25g trypsase powder to add in 100mL PBS solutions, add 0.02gEDTA, be placed in 4
Stirring at low speed 0.5h in DEG C ice bath, adjusts pH to 7.4, filtering packing, is placed in -20 DEG C of preservations.
The present invention is described in further detail with reference to following examples.It should be noted that in following examples
Described RPMI-1640 culture mediums are formulated using described in above-mentioned A, and " also adding " refers to the RPMI-1640 trainings described in A
It is added on the basis of foster base.RPMI-1640 culture mediums and phosphate buffer described in following examples are using upper
State formula.
Embodiment 1
A kind of method for separating and preparing of people's primary tumor cell, comprises the following steps：
S1：The separation of primary tumor cell：Tumor tissues are gathered, are cleaned up, slough and connective tissue is removed,
And cut into tissue pieces；
S2：First cultivation stage：The tissue pieces are inoculated in Tissue Culture Flask, 1mL tire containing low concentration is added
The RPMI-1640 culture mediums of cow's serum, are cultivated 2 weeks；
S3：Second cultivation stage：The RPMI-1640 culture mediums containing high concentration hyclone are changed, continue to cultivate to thin
Born of the same parents' degrees of fusion reaches 70~80%；
S4：Cell harvesting and freeze：Obtained cell is subjected to digestion harvest, frozen stock solution is added into the cell of harvest,
And frozen.
It should be noted that the scope of the low concentration hyclone described in the present embodiment is 40~60mg/L, it is described
The scope of high concentration hyclone is 80~120mg/L.
Embodiment 2
A kind of method for separating and preparing of people's primary tumor cell, comprises the following steps：
S1：The separation of primary tumor cell：Tumor tissues are gathered, are cleaned up, slough and connective tissue is removed,
And cut into tissue pieces；
S2：First cultivation stage：The tissue pieces are inoculated in Tissue Culture Flask, 5mL tire containing low concentration is added
The RPMI-1640 culture mediums of cow's serum, are cultivated 3 weeks；
S3：Second cultivation stage：The RPMI-1640 culture mediums containing high concentration hyclone are changed, continue to cultivate to thin
Born of the same parents' degrees of fusion reaches 70~80%；
S4：Cell harvesting and freeze：Obtained cell is subjected to digestion harvest, frozen stock solution is added into the cell of harvest,
And frozen.
Embodiment 3
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 1, wherein step S1's
Specific method is as follows：
Tumor tissues are gathered, are transferred under aseptic conditions in culture dish, with the sodium chloride that 5mL mass fractions are 0.9%
The aqueous solution is rinsed, and is repeated 3 times, and removes bloodstain, and cuts off slough and connective tissue, is cut into 1mm3Tissue pieces.
Embodiment 4
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 2, wherein step S1's
Specific method is as follows：
Tumor tissues are gathered, are transferred under aseptic conditions in culture dish, with the chlorination that 10mL mass fractions are 0.9%
Sodium water solution is rinsed, and is repeated 2 times, and removes bloodstain, and cuts off slough and connective tissue, is cut into 1mm3Tissue pieces.
Embodiment 5
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 3, wherein step S2's
Specific method is as follows：
S2.1：Tissue pieces are inoculated in T25 blake bottles, the RPMI-1640 of 1mL hyclones containing 50mg/L is added
Culture medium, tissue block is uniformly laid on blake bottle bottom, is placed in 37 DEG C of CO2Cultivated in incubator；
S2.2：A full dose was carried out every 3~4 days and changes liquid, old culture medium is discarded, while adding the fresh cultured of equivalent
Base, is placed in CO2Continue to cultivate in incubator；
S2.3：After culture 7~10 days, blake bottle is jiggled, tissue block is come off；By old culture medium and the group come off
The membrane filtration that block shifts and uses 100 μm in the lump is knitted, filtrate is collected, 5min is centrifuged under the conditions of 1500rpm/min, collect
Precipitation, adds new culture medium, continues to cultivate 1~2 week.
Embodiment 6
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 4, wherein step S2's
Specific method is as follows：
S2.1：Tissue pieces are inoculated in T25 blake bottles, the RPMI-1640 of 5mL hyclones containing 50mg/L is added
Culture medium, tissue block is uniformly laid on blake bottle bottom, is placed in 37 DEG C of CO2Cultivated in incubator；
S2.2：A full dose was carried out every 3~4 days and changes liquid, old culture medium is discarded, while adding the fresh cultured of equivalent
Base, is placed in CO2Continue to cultivate in incubator；
S2.3：After culture 7~10 days, blake bottle is jiggled, tissue block is come off；By old culture medium and the group come off
The membrane filtration that block shifts and uses 100 μm in the lump is knitted, filtrate is collected, 5min is centrifuged under the conditions of 2000rpm/min, collect
Precipitation, adds new culture medium, continues to cultivate 1~2 week.
S3：Second cultivation stage：The RPMI-1640 culture mediums containing high concentration hyclone are changed, continue to cultivate to thin
Born of the same parents' degrees of fusion reaches 70~80%；
S4：Cell harvesting and freeze：Obtained cell is subjected to digestion harvest, frozen stock solution is added into the cell of harvest,
And frozen.
Embodiment 7
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 5, wherein step S3's
Specific method is as follows：
S3：After first cultivation stage culture 2~3 weeks, old culture medium is discarded, is used instead containing 100mg/L hyclones
RPMI-1640 culture mediums, carry out a full dose in every 3~4 days and change liquid, cultivate untill when cell fusion degree reaches 70~80%.
Embodiment 8
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 6, wherein step S3's
Specific method is as follows：
S3：After first cultivation stage culture 2~3 weeks, old culture medium is discarded, is used instead containing 100mg/L hyclones
RPMI-1640 culture mediums, carry out a full dose in every 3~4 days and change liquid, cultivate untill when cell fusion degree reaches 70~80%.
Embodiment 9
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 7, wherein step S4's
Specific method is as follows：
S4.1：When cell total amount reaches 1*107When, old culture medium is discarded, is cleaned with phosphate buffer 2 times, is discarded and wash
Wash liquid；
S4.2：5mL 0.125% trypsase-EDTA digestive juices are added into the blake bottle after washing, are gently shaken
Digestive juice is infiltrated blake bottle bottom surface, stood in superclean bench after 1min, take out blake bottle and gently pat, be placed in down
Micro- Microscopic observation is put, untill when cell rounding, most cells come off；
S4.3：RPMI-1640 culture mediums containing high concentration hyclone described in adding 3ml into blake bottle, will be cultivated
The suspension transfer of cell and tissue block, is eluted the cell sticked in blake bottle with phosphate buffer described in 5mL in bottle,
And mix the residual cells of elution with the suspension, 5min is centrifuged under conditions of 1500rpm/min；
S4.4：Cell is resuspended in supernatant discarding after centrifugation, phosphate buffer described in addition 5mL, and gently piping and druming is mixed,
The cell suspension after resuspension is centrifuged into 5min, and supernatant discarding under conditions of 1500rpm/min；
S4.5：1mL frozen stock solutions are added into the cell being collected into, is transferred in cryopreservation tube, is put into program temperature reduction box, put
In -80 DEG C of refrigerator freezings, cryopreservation tube is transferred in liquid nitrogen and preserved by next day.
Embodiment 10
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 8, wherein step S4's
Specific method is as follows：
S4.1：When cell total amount reaches 1*107When, old culture medium is discarded, is cleaned with phosphate buffer 3 times, is discarded and wash
Wash liquid；
S4.2：1mL 0.25% trypsase-EDTA digestive juices are added into the blake bottle after washing, gently shaking makes
Digestive juice infiltration blake bottle bottom surface, stands after 1min in superclean bench, takes out blake bottle and gently pats, and is placed in being inverted
Micro- Microscopic observation, untill when cell rounding, most cells come off, overall process is no more than 5min；
S4.3：RPMI-1640 culture mediums containing high concentration hyclone described in adding 5ml into blake bottle, will be cultivated
The suspension transfer of cell and tissue block, is eluted the cell sticked in blake bottle with phosphate buffer described in 10mL in bottle,
And mix the residual cells of elution with the suspension, 5min is centrifuged under conditions of 2000rpm/min；
S4.4：Cell is resuspended in supernatant discarding after centrifugation, phosphate buffer described in addition 10mL, and gently piping and druming is mixed,
The cell suspension after resuspension is centrifuged into 5min, and supernatant discarding under conditions of 2000rpm/min；
S4.5：2mL frozen stock solutions are added into the cell being collected into, is transferred in cryopreservation tube, is put into program temperature reduction box, put
In -80 DEG C of refrigerator freezings, cryopreservation tube is transferred in liquid nitrogen and preserved by next day.
Embodiment 11
A kind of method for separating and preparing of people's primary tumor cell, including each step, wherein RPMI-1640 described in embodiment 9
Following composition is also added with culture medium：150mg/L Sodium Pyruvate and the dense element of 2mg/L mandarin orange.
Embodiment 12
A kind of method for separating and preparing of people's primary tumor cell, including each step, wherein RPMI- described in embodiment 10
Following composition is also added with 1640 culture mediums：The dense element of 100mg/L Sodium Pyruvate, 1mg/L mandarin orange, 6mg/L Y-27632
And 4mg/L griseofulvin.
Embodiment 13
A kind of method for separating and preparing of people's primary tumor cell, including each step, wherein RPMI-1640 described in embodiment 9
Following composition is also added with culture medium：150mg/L Sodium Pyruvate, 1mg/L mandarin orange it is dense element, 4mg/L Y-27632 and
2mg/L griseofulvin.
Embodiment 14
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 9, wherein frozen stock solution bag
Include following composition：Volume parts ratio is 5:4:1 hyclone, plant source recombination human serum albumin and dimethyl sulfoxide (DMSO).
Embodiment 15
A kind of method for separating and preparing of people's primary tumor cell, including each step described in embodiment 10, wherein frozen stock solution bag
Include following composition：Volume parts ratio is 7:2:1 hyclone, plant source recombination human serum albumin and dimethyl sulfoxide (DMSO).
Reference examples 1
A kind of method for separating and preparing of people's primary tumor cell, comprises the following steps：
S1：The separation of primary tumor cell：Tumor tissues are gathered, are cleaned up, slough and connective tissue is removed,
And cut into tissue pieces；
S2：Cultivation stage：The tissue pieces are inoculated in Tissue Culture Flask, the 1mL ox blood of tire containing 50mg/L is added
Clear RPMI-1640 culture mediums, carry out a full dose in every 3~4 days and change liquid, cultivate 3~4 weeks, 70 are reached to cell fusion degree~
80%；
S3：Cell harvesting and freeze：Obtained cell is subjected to digestion harvest, frozen stock solution is added into the cell of harvest,
And frozen.
Reference examples 2
A kind of method for separating and preparing of people's primary tumor cell, comprises the following steps：
S1：The separation of primary tumor cell：Tumor tissues are gathered, are cleaned up, slough and connective tissue is removed,
And cut into tissue pieces；
S2：Cultivation stage：The tissue pieces are inoculated in Tissue Culture Flask, the 1mL ox of tire containing 100mg/L is added
The RPMI-1640 culture mediums of serum, carry out a full dose in every 3~4 days and change liquid, cultivate 3~4 weeks, 70 are reached to cell fusion degree
~80%；
S3：Cell harvesting and freeze：Obtained cell is subjected to digestion harvest, frozen stock solution is added into the cell of harvest,
And frozen.
Experimental example 1
Primary cell propagation efficiency comparative's experiment
Respectively in the method for embodiment 1,2,7,8,9,10,11,12,13 as experimental group 1~9, the method for reference examples 1,2
For control group 1~2, carry out original cuiture to the cell of same tumor tissues, and compare each group cell quantity reaching 1*107When
The required time, and culture 4 weeks when each group cell viability.As a result it is as shown in table 1.
The each group cell culture time of table 1 compares
As shown in Table 1, the incubation time of experimental group each group is considerably less than control group, and cell viability is also significantly high
In control group, illustrate that the cultural method of the invention provided can effectively shorten cultivation cycle, raising cell yield particularly living thin
The yield of born of the same parents.Wherein, the incubation time of experimental group 5~7 is most short, cell viability highest, shows to change culture medium in the present invention
Enter to significantly improve the multiplication rate and activity of cell.
Experimental example 2
Cell surface immunophenotype genetic experiment
Respectively in the method for embodiment 1,2,11,12,13 as experimental group 1~5, with conventional tissue mass cell culture and enzyme
Method as a control group 1~2, is cultivated tumour cell, and carries out flow cytometer detection to the surface antigen of the cell of acquisition, together
When positive control is used as using mother cell.As a result it is as shown in table 2.
The each group cell surface antigen flow cytometer detection result of table 2
As shown in Table 2, experimental group and control group each group are compared with positive control, and cell surface immunophenotype species is homogeneous
Together, and each cell surface antigen ratio is not significantly different.It can thus be appreciated that by the cell and former tumor tissues of original cuiture
Cell has identical surface antigen, illustrates that the tumour cell of primary culture in vitro has reproduced interior tumor cell well
Genetic phenotype, genetic stability is good.
Experimental example 3
Frozen stock solution preservation effect is tested
The frozen stock solution using the offer of embodiment 14~15 is as experimental group 1~2 respectively, with (the 90% tire ox of conventional cryopreservation liquid 1
Serum+10%DMSO) and conventional cryopreservation liquid 2 (dimethyl sulfoxide (DMSO) of 95% complete culture solution+5%) as a control group 1~2, it is right
The method provided by embodiment 13 carries out the obtained same a collection of cell of original cuiture and frozen respectively, respectively at 7d, 14d,
28d determines the cell viability of each group and is compared.As a result it is as shown in table 3.
The each group cell viability situation of change of table 3 is contrasted
As shown in Table 3, the cell viability variation tendency of experimental group each group is close with control group 1, and reduction amplitude is very in 28d
It is small, and the cell viability during freezing of control group 2 is significantly reduced.In use, it is generally recognized that serum content is higher, freeze
Liquid storage nutrition is abundanter, for maintaining the effect of cell viability better.Plant source human seralbumin egg is used in experimental group each group
Come partly to replace hyclone in vain, still freeze effect with good, while the consumption of serum, reduction behaviour can also be reduced
Make cost.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the present invention's
Protection domain.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (9)
1. a kind of method for separating and preparing of people's primary tumor cell, it is characterised in that comprise the following steps：
S1：The separation of primary tumor cell：Tumor tissues are gathered, are cleaned up, slough and connective tissue are removed, and cut
Into tissue pieces；
S2：First cultivation stage：The tissue pieces are inoculated in Tissue Culture Flask, the 1~5mL ox of tire containing low concentration is added
The RPMI-1640 culture mediums of serum, are cultivated 2~3 weeks；
S3：Second cultivation stage：The RPMI-1640 culture mediums containing high concentration hyclone are changed, continues to cultivate to cell and melts
It is right to reach 70~80%；
S4：Cell harvesting and freeze：Obtained cell is subjected to digestion harvest, frozen stock solution is added into the cell of harvest, goes forward side by side
Row freezes.
2. the method for separating and preparing of people's primary tumor cell as claimed in claim 1, it is characterised in that the tool of the step S1
Body method is as follows：
Tumor tissues are gathered, are transferred under aseptic conditions in culture dish, with the sodium chloride that 5~10mL mass fractions are 0.9%
The aqueous solution is rinsed, and is repeated 2~3 times, removes bloodstain, and cuts off slough and connective tissue, is cut into 1mm3Tissue pieces.
3. the method for separating and preparing of people's primary tumor cell as claimed in claim 1, it is characterised in that the step S2 includes
Following steps：
S2.1：Tissue pieces are inoculated in T25 blake bottles, the RPMI-1640 trainings of 1~5mL hyclones containing 50mg/L are added
Base is supported, tissue block is uniformly laid on to blake bottle bottom, 37 DEG C of CO is placed in2Cultivated in incubator；
S2.2：A full dose was carried out every 3~4 days and changes liquid, old culture medium is discarded, while adding the fresh culture of equivalent, was put
In CO2Continue to cultivate in incubator；
S2.3：After culture 7~10 days, blake bottle is jiggled, tissue block is come off；By old culture medium and the tissue block one come off
And 100 μm of membrane filtration is shifted and uses, filtrate is collected, 5min is centrifuged under the conditions of 1500~2000rpm/min, collects heavy
Form sediment, add new culture medium, continue to cultivate 1~2 week.
4. the method for separating and preparing of people's primary tumor cell as claimed in claim 1, it is characterised in that the tool of the step S3
Body method is as follows：
After first cultivation stage culture 2~3 weeks, old culture medium is discarded, the RPMI-1640 containing 100mg/L hyclones is used instead
Culture medium, carries out a full dose in every 3~4 days and changes liquid, cultivate untill when cell fusion degree reaches 70~80%.
5. the method for separating and preparing of people's primary tumor cell as claimed in claim 1, it is characterised in that the step S4 includes
Following steps：
S4.1：When cell total amount reaches 1*107When, old culture medium is discarded, is cleaned with phosphate buffer 2~3 times, discards washing
Liquid；
S4.2：1~5mL 0.125~0.25% trypsase-EDTA digestive juices are added into the blake bottle after washing, gently
Shake makes digestive juice infiltrate blake bottle bottom surface, is stood in superclean bench after 1min, takes out blake bottle and gently pats, is placed in
Observed under inverted microscope, untill when cell rounding, most cells come off；
S4.3：The RPMI-1640 culture mediums containing high concentration hyclone described in 3~5ml are added into blake bottle, by blake bottle
The suspension transfer of middle cell and tissue block, is eluted the cell sticked in blake bottle with phosphate buffer described in 5~10mL,
And mix the residual cells of elution with the suspension, 5min is centrifuged under conditions of 1500~2000rpm/min；
S4.4：Cell is resuspended in supernatant discarding after centrifugation, phosphate buffer described in 5~10mL of addition, and gently piping and druming is mixed,
The cell suspension after resuspension is centrifuged into 5min, and supernatant discarding under conditions of 1500~2000rpm/min；
S4.5：1~2mL frozen stock solutions are added into the cell being collected into, is transferred in cryopreservation tube, is put into program temperature reduction box, be placed in-
Cryopreservation tube is transferred in liquid nitrogen and preserved by 80 DEG C of refrigerator freezings, next day.
6. such as the method for separating and preparing of people's primary tumor cell according to any one of claims 1 to 5, it is characterised in that institute
State RPMI-1640 culture mediums and be also added with following composition：100~150mg/L Sodium Pyruvate and the dense element of 1~2mg/L mandarin orange.
7. the method for separating and preparing of people's primary tumor cell as claimed in claim 6, it is characterised in that the RPMI-1640
Following composition is also added with culture medium：4~6mg/L Y-27632 and 2~4mg/L griseofulvin.
8. the method for separating and preparing of people's primary tumor cell as claimed in claim 5, it is characterised in that the frozen stock solution includes
Following composition：Volume parts ratio is 5~7:2~4:1 hyclone, plant source recombination human serum albumin and dimethyl is sub-
Sulfone.
9. the method for separating and preparing of people's primary tumor cell as claimed in claim 5, it is characterised in that the step S4.2's
Duration is no more than 5min.
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