CN104388382B - Method for rapidly preparing Feeder cells by using stirring-type cell culturing bottle rolling machine - Google Patents
Method for rapidly preparing Feeder cells by using stirring-type cell culturing bottle rolling machine Download PDFInfo
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Abstract
The invention discloses a method for rapidly preparing of Feeder cells by using a stirring-type cell culturing bottle rolling machine. The method comprises the three steps of the primary acquisition of CF-1 MEFs, the subculturing of CF-1 MEFs and the preparation of a CF-1 MEF Feeder. During the preparation of the CF-1 MEF Feeder, the stirring-type cell culturing bottle rolling machine serves as cell processing equipment, and a stirring hammer of the stirring-type cell culturing bottle rolling machine rotates constantly under the action of an electromagnetic field, so that cells can fully contact MMC in a culturing solution in a suspension state, and thus the efficiency of inhibiting the proliferation of the cells by using the MMC can be improved, and the cell processing time can be shortened. The special culturing bottle of the stirring-type cell culturing bottle rolling machine is several or hundreds of times the volume of a common culturing bottle/utensil and can be used repeatedly, and thus a large number of culturing bottles/utensils can be saved, the preparation cost of the Feeder can be reduced, the preparation efficiency of the Feeder can be improved, and a lot of time spent in doing experiments can be saved. In addition, according to the method, the cells are processed in a suspension state, and the cells do not need to be treated by using a digestive solution when being recovered, and thus the damage to the cells caused by digestion can be alleviated, and the cells can be kept in good states to the maximum extent.
Description
Technical field
The present invention relates to a kind of preparation method of feeder layer (Feeder) cell, and in particular to one kind utilizes stirring-type cell
The method that culture Rotary Machine quickly prepares feeder cells, belongs to Cell culture invitro technical field.
Background technology
Induction type pluripotent stem cell（Induced pluripotent stem cells, iPSCs）Many characteristics and embryo
Tire stem cell is very much like, can be divided into almost all of adult cell, because it can reprogram technology from whole end by cell
The adult cell of differentiation is obtained, therefore can obtain patient-specific or disease specific iPSCs, promotes it as study of disease
The focus of mechanism, Individual drug treatment, drug screening and cell therapy, further furthered stem cell and clinical disease
The distance for the treatment of.IPSCs is susceptible to differentiation and the unstable situation of caryogram during long-term cultivation, during long-term cultivation
The normal undifferentiated states of iPSCs and self-renewal capacity is maintained to be an extremely challenging job.And long-term cultivation is being raised
It is to maintain the undifferentiated states of iPSCs and caryogram stably most simple, direct and effective method in coating systems, therefore prepares high-quality
Feeder become culture iPSCs key technology.
At present, Feeder preparation methods mainly have gamma-radiation irradiation and mitomycin（Mitomycin C, MMC）Process
Method：The advantage of gamma-rays method be can mass disposal cell, but due to gamma-rays exist radioactive pollution and storage supervision journey
Sequence is loaded down with trivial details, and the shortcomings of somewhat expensive, in the world many areas can not find radioactive sources, therefore most experiments research still using MMC
Method is preparing Feeder.MEC（Mouse embryonic fibroblasts, MEFs）, it is the most frequently used
Feeder cell deriveds；And CF-1 MEFs are culture people iPSCs（hiPSCs）The most frequently used Feeder cell deriveds, have
Multiplication capacity is extremely strong, can coating growth, density dependency high and low density when the features such as easily occur aging.
When traditional MMC methods prepare CF-1 MEF Feeder, it usually needs by MEFs spreading to cell culture compared with low-density
In ware/bottle, treat that it is grown to≤100% fusion（Ensure cell for individual layer presence）When, suppress its mitosis with MMC, exist following
Problem：1) yield poorly, high cost：Cell quantity using conventional method Single cell culture ware/bottle process is less, to expect
Sufficient amount of Feeder is accomplished by consuming substantial amounts of experiment consumptive material, and many batch processings necessarily lead to the expense of costliness； 2）Cell
Proliferation Ability is insufficient：If improving the density that Single cell culture ware/bottle processes cell, it may appear that cell propagation can not be abundant
The problem of suppression；3) the biological behaviour susceptible of MEFs：Easily there is aging, secretion during due to CF-1 MEFs low-density
HiPSCs must the ability of trophic factors will decline, the growth conditions of hiPSCs may be had influence on；4）Process time is limited：For
Abundant suppression cell propagation, with MMC process preferably when cell growth is to 80% ~ 90% fusion, but CF-1 MEFs growth rates
Quickly, easily there is coating growth so as to miss optimization process opportunity.In order to reduce the preparation cost of Feeder, improve cell and process
Efficiency, reduces operator's working strength, is badly in need of a kind of new method, both can a large amount of cells of single batch processing, can guarantee that cell increases again
Grow and be adequately suppressed.
The content of the invention
The technical problem to be solved is for the deficiencies in the prior art, there is provided a kind of to be trained using stirring-type cell
The method that foster Rotary Machine quickly prepares Feeder cells, using stirring-type red blood-cell cultivation rotating-bottle machine as cell processing apparatus, stirring
Hammer can constantly rotate under electromagnetic field effect, cell is fully contacted with the MMC in nutrient solution under suspended state, can improve
MMC suppresses the efficiency of cell propagation, shortens process time；Cell is processed under suspended state, is disappeared without the need for digestive juice when reclaiming cell
Change, the damage that digestion brings to cell can be reduced, the kilter of cell is kept to greatest extent；The volume of Tissue Culture Flask is
The even hundreds of times of several times of Nostoc commune Vanch bottle/ware, can process a large amount of cells in the short time, can improve the preparation efficiency of Feeder,
The preparation cost of Feeder is reduced, is operated more flexible.
In order to achieve the above object, the technical solution used in the present invention is as follows：
A kind of method that utilization stirring-type red blood-cell cultivation rotating-bottle machine quickly prepares Feeder cells, including CF-1 MEFs are former
Three steps such as generation acquisition, CF-1 MEFs Secondary Cultures and CF-1 MEF Feeder preparations, it is characterised in that prepare CF-1
MEF Feeder specifically include following steps using stirring-type red blood-cell cultivation rotating-bottle machine as cell processing apparatus：
（1）CF-1 MEFs primary (P0) are obtained：
I, the CF-1 mouse for taking pregnant 12.5 days, under de- neck execution, aseptic condition embryo is taken out, and uses 10ml PBS（That is phosphoric acid
Salt buffer）It is put in sterile petri dish after cleaning；Embryo's head and internal organ are removed, with 10ml PBS 2 times are cleaned；
II, remaining tissue is moved in new sterile petri dish, shredded in tissue with sterile scissors, each embryo adds
1ml digestive juices, while piping and druming is for several times, is put in incubator and digests 5min；Add and terminate disappearing with the isopyknic nutrient solution of digestive juice
Abundant piping and druming after change is separated into unicellular as far as possible；
III, above-mentioned cell suspension is mixed after, each T75 blake bottle adds the cell suspension and 13ml culture of 2 embryos
Liquid, then blake bottle is put in incubator cultivate, and on blake bottle Cell Name, generation P0 and date are indicated.
（2）CF-1 MEFs Secondary Cultures：
1. after, P0 is cultivated 3 days for CF-1 MEFs, when observation MEFs covers with blake bottle bottom under inverted microscope, culture is abandoned in suction
Liquid, cell one time is cleaned with 10ml PBS, is subsequently adding 2ml digestive juices and is mixed to covering whole blake bottle bottom；
2., blake bottle is placed in incubator and is incubated 3 minutes, and patting blake bottle side wall with hand makes MEFs cell detachments；Use 2ml
Nutrient solution terminates digestion, while gently piping and druming forms single cell suspension；
3., above-mentioned cell suspension is assigned in 3 T75 bottles（As 1:3 Secondary Cultures）, mend to 15ml with nutrient solution
Row is passed on, and is put in incubator and is continued to cultivate；
, use the method, cell was reached into for the 3rd generation.
（3）, CF-1 MEF Feeder prepare：
A：To be placed in incubator after the magnetic stirring apparatus sterilization of stirring-type red blood-cell cultivation rotating-bottle machine, magnetic stirring apparatus passes through
Control line is connected with the control cabinet outside incubator；It is cold after the special Tissue Culture Flask and stirring hammer high temperature, autoclaving of Rotary Machine
But it is stand-by to room temperature；
B, CF-1 MEFs was cultivated to the 3rd the 4th day generation, and nutrient solution is abandoned in suction, with 10ml PBSs cell one time, then
2ml digestive juices are added to digest in incubator 3 minutes, patting blake bottle side wall with hand makes cell detachment, adds 2ml nutrient solution ends
Only digest, count after cell is collected in same centrifuge tube；
C, the cell collected in step B is transferred in special Tissue Culture Flask, according to cell number add nutrient solution to 25 ~
1000ml, cell density is adjusted to 0.5 × 106~1.3×106Individual/ml；Then MMC is added to into above-mentioned culture under the conditions of lucifuge
In liquid, MMC concentration is adjusted to 10 g/ml, after fully mixing bottle cap is screwed；
On D, special Tissue Culture Flask is placed in incubator magnetic stirring apparatus immediately, bottle cap is unscrewed, by control cabinet
Rotation mode is set to 10 ~ 20 revs/min, continuously rotates 0.5 ~ 2 hour；The outstanding pine of lid of the special Tissue Culture Flask of the present invention,
Both the sterile culture condition of cell had been can ensure that, the necessary oxygen demand of cells survival can have been met again, while the CO in incubator2
Can also enter in blake bottle and adjust the pH of internal media；
After E, rotation terminate, cell is transferred in 50ml centrifuge tubes immediately, is put in centrifuge with 1000 revs/min
Centrifugation 5 ~ 15 minutes, then inhales and abandons supernatant in centrifuge tube, removes the nutrient solution containing MMC；
F, PBS re-suspended cells are used, cell density is adjusted to into 2 × 105~5×105Individual/ml, with 1000 revs/min of centrifugations 5
~ 15 minutes, after centrifugation terminates, supernatant was abandoned in suction；
G, it is repeated 3 times by step F methods described, obtains the cell for needing, it is frozen standby after cell count.
Preferably, the stirring-type red blood-cell cultivation rotating-bottle machine described in step A is Cell Spin than Europe cell-culturing rotating bottle
Machine--4 bottles of stirring-type position.
Preferably, in step C, cell density is adjusted to 0.6 × 106~1×106Individual/ml；In step F, cell density adjustment
To 3 × 105~4×105Individual/ml.
Preferably, in step D, rotation mode on control cabinet is set to into 15 revs/min, is continuously rotated 0.5 ~ 1.5 hour；
In step E, with 1000 revs/min of centrifugation 10 minutes, the cell loss caused because of centrifugation is reduced as far as possible, while can
Avoid because centrifugation time is long and/or the excessive impact cell state of rotating speed.
Preferably, in step G, each component volume ratio of frozen formula of liquid is nutrient solution：Hyclone（FBS）：Dimethyl is sub-
Sulfone（DMSO）=9:10:1.
Preferably, nutrient solution of the present invention is：With DMEM in high glucose nutrient solution as basic culture solution, to basic culture solution
Middle addition volume fraction is 10% hyclone, 1% nonessential amino acid, 1% Glu, is used after fully mixing
It is standby after 0.22 m filter filtration sterilizations；Described digestive juice is 0.25% pancreatin+0.02%EDTA.
The method that utilization stirring-type red blood-cell cultivation rotating-bottle machine of the present invention quickly prepares Feeder cells, the method is also
Can apply to field of cell culture other cells preparation Feeder.
The inventive method, the stirring hammer of stirring-type red blood-cell cultivation rotating-bottle machine can constantly rotate under electromagnetic field effect, make thin
Born of the same parents are fully contacted under suspended state with the MMC in nutrient solution, can improve the efficiency that MMC suppresses cell propagation, when shortening process
Between；The volume of Rotary Machine special culture bottle has 100ml, 250ml, 500ml, 1000ml different size, and blake bottle can be reused,
A large amount of Tissue Culture Flask/wares can be saved, the preparation cost of Feeder is reduced；The a large amount of cells of short time interior energy process, can improve
Feeder preparation efficiencies, save many experiments time, operate more flexible, hence it is evident that reduce the working strength of operator；Separately
Outer this method is that cell is processed under suspended state, is digested without the need for digestive juice when reclaiming cell, can reduce digestion and cell is brought
Damage, to greatest extent keep cell kilter.
Description of the drawings
Fig. 1：According to Feeder prepared by the method for embodiment 1, MMC processes the aobvious of the Feeder cells that obtain after different time
Micro-structural figure；
Fig. 2：According to Feeder prepared by the method for embodiment 1, MMC is processed after different time, the time dependent increasing of cell
Grow statistics；
Fig. 3：According to Feeder prepared by the method for embodiment 1, MMC is processed after different time, the anabiosis rate statistics knot of cell
Really；
Fig. 4：The Feeder prepared after different time is processed according to the method for embodiment 1, MMC, after inoculation hiPSCs, Feeder
The micro-structure diagram of cell and culture hiPSCs thereon；
Fig. 5：According to Feeder prepared by the method for embodiment 2, MMC processes the aobvious of the Feeder cells that obtain after different time
Micro-structural figure；
Fig. 6：According to Feeder prepared by the method for embodiment 2, MMC is processed after different time, the time dependent increasing of cell
Grow statistics；
Fig. 7：According to Feeder prepared by the method for embodiment 2, MMC is processed after different time, the anabiosis rate statistics knot of cell
Really；
Fig. 8：The Feeder prepared after different time is processed according to the method for embodiment 2, MMC, after inoculation hiPSCs, Feeder
The micro-structure diagram of cell and culture hiPSCs thereon；
Wherein：
In Fig. 1, Fig. 4, Fig. 5, Fig. 8：A, B, C, D represent respectively MMC and process 0.5h, 1h, 1.5h, 2h；
In Fig. 2:Represent the 1st day,Represent the 3rd day,Represent the 5th day,Represent the 7th day；
In Fig. 6:Represent the 1st day,Represent the 3rd day,Represent the 5th day,Represent the 7th day.
Specific embodiment
The present invention is specifically described with reference to specific embodiment and embodiment.
Embodiment 1：
A kind of method that utilization stirring-type red blood-cell cultivation rotating-bottle machine quickly prepares Feeder cells, comprises the following steps：
（1）CF-1 MEFs are primary（P0）Obtain：
The CF-1 mouse of pregnant 12.5 days are taken, de- neck is put to death, take out embryo under aseptic condition, with being put in after 10ml PBSs
In sterile petri dish；The head and internal organ of embryo are removed, with 10ml PBS 2 times are cleaned；Remaining tissue is transferred to new aseptic
Shredded with sterile scissors in culture dish, by each embryo 1ml digestive juice is added, while piping and druming is for several times, be put into 37 DEG C, 5%CO2Carefully
5min is digested in born of the same parents' incubator；Terminate digestion with the isopyknic nutrient solution of digestive juice, fully piping and druming is separated into unicellular as far as possible；
After above-mentioned cell suspension is mixed, the cell suspension and 13ml nutrient solutions of 2 embryos is added in each T75 blake bottle, it is fully mixed
Blake bottle is put in incubator after even P0 is cultivated for cell.
（2）CF-1 MEFs Secondary Cultures：
After P0 is cultivated 3 days for MEFs, MEFs is observed under inverted microscope and covers with blake bottle bottom, suction is abandoned nutrient solution, uses 10ml
PBS cleanings cell one time, is subsequently adding 2ml digestive juices and digests in incubator 3 minutes, and patting side wall with hand makes cell detachment；
Terminated digesting with 2ml nutrient solutions, while gently piping and druming forms single cell suspension；By cell with 1:3 ratio carries out Secondary Culture
To the 3rd generation.
（3）, CF-1 MEF Feeder prepare：
A：To be placed in incubator after the magnetic stirring apparatus sterilization of stirring-type red blood-cell cultivation rotating-bottle machine, magnetic stirring apparatus passes through
Control line is connected with the control cabinet outside incubator；Volume hammers high temperature, height into shape for the special Tissue Culture Flask of Rotary Machine of 100ml with stirring
It is stand-by room temperature to be cooled to after pressure sterilizing；
B, step（2）In CF-1 MEFs cultivate to the 3rd the 4th day generation, suction abandon nutrient solution, it is thin with 10ml PBSs
Born of the same parents one time, are subsequently adding 2ml digestive juices and digest in incubator 3 minutes, and patting side wall with hand makes cell detachment, is cultivated with 2ml
Liquid terminates digestion, counts after cell is collected in same centrifuge tube；
C, the cell collected by step B is transferred in special Tissue Culture Flask, according to cell number add nutrient solution to 50 ~
100ml, cell density is adjusted to 0.6 × 106~1×106Individual/ml；Then MMC is added to into above-mentioned nutrient solution under the conditions of lucifuge
In, MMC concentration is adjusted to 10 g/ml, and after fully mixing bottle cap is screwed；
On D, special Tissue Culture Flask is placed in incubator magnetic stirring apparatus immediately, bottle cap is unscrewed, by control cabinet
Rotation mode is set to 15 revs/min, removes blake bottle after 0.5h, 1h, 1.5h, 2h respectively；
E, it is rapid the cell that the different rotary time obtains is transferred to respectively in 50ml centrifuge tubes, be put in centrifuge with
1000 revs/min of centrifugation 10 minutes, inhales abandon supernatant in centrifuge tube respectively, removes the nutrient solution containing MMC；
F, PBS re-suspended cells are used, cell density is adjusted to into 3 × 105Individual/ml, is centrifuged 10 minutes with 1000 revs/min,
After centrifugation terminates, supernatant is abandoned in suction；
G, it is repeated 3 times by step F methods described, obtains the cell for needing, it is frozen standby after cell count.
0.5h is processed using stirring-type red blood-cell cultivation rotating-bottle machine MMC（A）、1h（B）、1.5h（C）、2h（D）The CF- for obtaining afterwards
1 MEF Feeder, with 1 × 104~1.5×104/cm2Density recovery after its micro-structure diagram it is as shown in Figure 1.
G1- detections test 1：
Cell before will be frozen in above-mentioned steps G presses 1 × 105The density in individual/hole is seeded in 24 orifice plates, respectively at the 1st,
3rd, cell count is carried out within 5,7 days, as a result as shown in Figure 2：The 1st, 3,5,7 days cell numbers of 0h groups substantially increase, and have notable statistics
Difference, illustrates that the CF-1 MEFs that unused MMC is processed can breed rapidly；MMC processes the CF-1 after 0.5h, 1h, 1.5h, 2h
MEFs in the 1st, 3,5,7 days cell numbers without substantially increase and decrease, illustrate using stirring-type red blood-cell cultivation rotating-bottle machine MMC process 0.5h,
CF-1 MEFs propagation after 1h, 1.5h, 2h is adequately suppressed, and cell can survive at least 7 days and occur without substantially dead
Die, it is sufficient to meet the requirement that 5 ~ 7 days pass on once when hiPSCs is normally cultivated.
G2- detections test 2：
By the cell recovery of the frozen 1st, 7,21,28 days in above-mentioned steps G into T25 bottles, estimation utilizes stirring-type cell
Culture Rotary Machine MMC processes the anabiosis rate of Feeder cells prepared by 0.5h, 1h, 1.5h, 2h, as a result as shown in Figure 3：Using stirring
Mixing the CF-1 MEF Feeder cell recoveries rates obtained after formula red blood-cell cultivation rotating-bottle machine MMC process 0.5h, 1h, 1.5h can reach
To 91% ~ 95%, and the CF-1 MEF Feeder anabiosis rates that MMC process 2h are obtained are only 80% ~ 83%, illustrate thin using stirring-type
The Feeder cells that born of the same parents culture Rotary Machine MMC process 0.5h ~ 1.5h are obtained are better than 2h.
G3- detections test 3：
By cell frozen in above-mentioned steps G with 2.3 × 104~3×104Individual/cm2Density recovery to diameter 10cm training
In foster ware, human embryo stem cell nutrient solution is changed to after cell attachment, is then inoculated with hiPSCs thereon, as a result as shown in Figure 4：
0.5h is processed using stirring-type red blood-cell cultivation rotating-bottle machine MMC（A）、1h（B）、1.5h（C）、2h（D）The CF-1 MEF for obtaining afterwards
Feeder can well support the growth of hiPSCs, and can maintain the undifferentiated growths of hiPSCs more than 10 generations.
By the inventive method, the blake bottle of a 100ml once just can process CF-1 MEFs about 1 × 108Individual cell,
And the density for processing cell according to traditional MMC methods is calculated, equivalent to the Tissue Culture Dish of 16 ~ 24 diameter 10cm；Present invention side
Method can improve Feeder preparation efficiencies, when saving many experiments relative to prior art, a large amount of cells of short time interior energy process
Between, operate more flexible, hence it is evident that reduce the working strength of operator；Cell is processed under suspended state, when reclaiming cell
Without the need for digestive juice digestion, the damage that digestion brings to cell can be reduced, the kilter of cell is kept to greatest extent.
Embodiment 2：
The method of operating of embodiment 2 is similar to Example 1, except that：
In step A, the volume of Tissue Culture Flask is 1000ml；
In step C, nutrient solution is added to 800ml according to cell number, cell density is adjusted to 1 × 106Individual/ml；
In step F, cell density is adjusted to 3.5 × 105Individual/ml;
0.5h is processed using stirring-type red blood-cell cultivation rotating-bottle machine MMC（A）、1h（B）、1.5h（C）、2h（D）The CF- for obtaining afterwards
1 MEF Feeder, with 1 × 104~1.5×104/cm2Density recovery after its micro-structure diagram it is as shown in Figure 5.
G1- detections test 1：
Cell before will be frozen in above-mentioned steps G presses 1 × 105The density in individual/hole is seeded in 24 orifice plates, respectively at the 1st,
3rd, cell count is carried out within 5,7 days, as a result as shown in Figure 6：The 1st, 3,5,7 days cell numbers of 0h groups substantially increase, and have notable statistics
Difference, illustrates that the CF-1 MEFs that unused MMC is processed can breed rapidly；MMC processes the CF-1 after 0.5h, 1h, 1.5h, 2h
MEFs in the 1st, 3,5,7 days cell numbers without substantially increase and decrease, illustrate using stirring-type red blood-cell cultivation rotating-bottle machine MMC process 0.5h,
CF-1 MEFs propagation after 1h, 1.5h, 2h is adequately suppressed, and cell can survive at least 7 days and occur without substantially dead
Die, it is sufficient to meet the requirement that 5 ~ 7 days pass on once when hiPSCs is normally cultivated.
G2- detections test 2：
By the cell recovery of the frozen 1st, 7,21,28 days in above-mentioned steps G into T25 bottles, estimation utilizes stirring-type cell
Culture Rotary Machine MMC processes the anabiosis rate of Feeder cells prepared by 0.5h, 1h, 1.5h, 2h, as a result as shown in Figure 7：Using stirring
Mixing the CF-1 MEF Feeder cell recoveries rates obtained after formula red blood-cell cultivation rotating-bottle machine MMC process 0.5h, 1h, 1.5h can reach
To 91% ~ 95%, and the CF-1 MEF Feeder anabiosis rates that MMC process 2h are obtained are only 80% ~ 83%, illustrate thin using stirring-type
The Feeder cells that born of the same parents culture Rotary Machine MMC process 0.5h ~ 1.5h are obtained are better than 2h.
G3- detections test 3：
By cell frozen in above-mentioned steps G with 2.3 × 104~3×104Individual/cm2Density recovery to diameter 10cm training
In foster ware, human embryo stem cell nutrient solution is changed to after cell attachment, is then inoculated with hiPSCs thereon, as a result as shown in Figure 8：
0.5h is processed using stirring-type red blood-cell cultivation rotating-bottle machine MMC（A）、1h（B）、1.5h（C）、2h（D）The CF-1 MEF for obtaining afterwards
Feeder can well support the growth of hiPSCs, and can maintain the undifferentiated growths of hiPSCs more than 10 generations.
By the inventive method, once most multipotency process MEFs is about 1 × 10 to the blake bottle of a volume 1000ml9It is individual thin
Born of the same parents, and the density for processing cell according to traditional MMC methods is calculated, equivalent to the Tissue Culture Dish of 160 ~ 240 diameter 10cm, can pole
The earth improves Feeder preparation efficiencies, saves many experiments time, hence it is evident that reduce the working strength of operator.
Examples detailed above is technology design to illustrate the invention and technical characterstic, can not limit the present invention's with this
Protection domain.Equivalent transformation or modification that all essence of the invention is done, all should cover in protection scope of the present invention
Within.
Claims (8)
1. a kind of method that utilization stirring-type red blood-cell cultivation rotating-bottle machine quickly prepares Feeder cells, including CF-1 MEFs are primary
Obtain, CF-1 MEFs Secondary Cultures and CF-1 MEF Feeder prepare three steps, it is characterised in that prepare CF-1 MEF
Using stirring-type red blood-cell cultivation rotating-bottle machine as cell processing apparatus during Feeder, the rotary hammer of Rotary Machine under electromagnetic field effect not
Disconnected rotation, so that cell is fully contacted under suspended state with the MMC in nutrient solution；Suspended state processes cell, reclaims thin
Without the need for digestive juice process during born of the same parents, the damage that digestion brings to cell is reduced；The rotary hammer of Rotary Machine is continuous under electromagnetic field effect
Rotation, its rotary speed is 10 ~ 20 revs/min, is continuously rotated 0.5 ~ 2 hour.
2. method according to claim 1, it is characterised in that reclaim cell and refer to without the need for digestive juice process and suspending
Cell is processed under state, after process terminates, cell is inhaled after being directly centrifuged and abandons supernatant, then with after PBS re-suspended cells precipitation
Cleaning, without digestive juice digestion process.
3. method according to claim 2, it is characterised in that prepare concretely comprising the following steps for CF-1 MEF Feeder：
A：To be placed in incubator after the magnetic stirring apparatus sterilization of stirring-type red blood-cell cultivation rotating-bottle machine, magnetic stirring apparatus is by control
Line is connected with the control cabinet outside incubator；It is cooled to after the special Tissue Culture Flask and stirring hammer high temperature, autoclaving of Rotary Machine
Room temperature is stand-by；
B, CF-1 MEFs was cultivated to the 3rd the 4th day generation, added 2ml digestive juices to digest in incubator 3 minutes, and with hand training is patted
Foster bottle side wall makes cell detachment, is then terminated digesting with 2ml nutrient solutions, counts after cell is collected in same centrifuge tube；
C, the cell collected in step B is transferred in special Tissue Culture Flask, according to cell number add nutrient solution to 25 ~
1000ml, cell density is adjusted to 0.5 × 106~1.3×106Individual/ml；Then MMC is added to into above-mentioned culture under the conditions of lucifuge
In liquid, MMC concentration is adjusted to 10 g/ml, after fully mixing bottle cap is screwed；
On D, special Tissue Culture Flask is placed in incubator magnetic stirring apparatus immediately, bottle cap is unscrewed, will be rotated on control cabinet
Pattern is set to 10 ~ 20 revs/min, continuously rotates 0.5 ~ 2 hour；
After E, rotation terminate, cell is transferred in 50ml centrifuge tubes immediately, is put in centrifuge with 1000 revs/min of speed
Centrifugation 5 ~ 15 minutes, after centrifugation terminates, supernatant in centrifuge tube is abandoned in suction, removes the nutrient solution containing MMC；
F, PBS re-suspended cells are used, cell density is adjusted to into 2 × 105~5×105Individual/ml, with 1000 revs/min of centrifugations 5 ~ 15
Minute, after centrifugation terminates, supernatant is abandoned in suction；
G, it is repeated 3 times by step F methods described, obtains required cell, it is frozen standby after cell count.
4. method according to claim 3, it is characterised in that the stirring-type red blood-cell cultivation rotating-bottle machine described in step A, be
Cell Spin are than Europe red blood-cell cultivation rotating-bottle machine--4 bottles of stirring-type position.
5. method according to claim 3, it is characterised in that cell density is adjusted to 0.6 × 10 in step C6~1×106
Individual/ml；Cell density is adjusted to 3 × 10 in step F5~4×105Individual/ml.
6. method according to claim 3, it is characterised in that rotation mode is set to 15 revs/min on control cabinet in step D
Clock, continuously rotates 0.5 ~ 1.5 hour；In step E and F, centrifuge is with 1000 revs/min of centrifugation 10 minutes.
7. method according to claim 3, it is characterised in that in step G, frozen stock solution each component volume ratio is nutrient solution:
Hyclone:Dimethyl sulfoxide (DMSO)=9:10:1.
8. method according to claim 3, it is characterised in that described nutrient solution is：With DMEM in high glucose nutrient solution as base
This nutrient solution, hyclone, 1% nonessential amino acid, 1% L- paddy that volume fraction is 10% are added in basic culture solution
Glutamine, with standby after 0.22 m filter filtration sterilizations after fully mixing；Described digestive juice is 0.25% pancreatin+0.02%
EDTA。
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| CN104388382B true CN104388382B (en) | 2017-05-10 |
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