CN102586107A - Method for recovering flaky carrier - Google Patents
Method for recovering flaky carrier Download PDFInfo
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- CN102586107A CN102586107A CN2012100386830A CN201210038683A CN102586107A CN 102586107 A CN102586107 A CN 102586107A CN 2012100386830 A CN2012100386830 A CN 2012100386830A CN 201210038683 A CN201210038683 A CN 201210038683A CN 102586107 A CN102586107 A CN 102586107A
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- chip carrier
- pure water
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- solution
- water
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- 238000000034 method Methods 0.000 title claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 86
- 102000004142 Trypsin Human genes 0.000 claims abstract description 35
- 108090000631 Trypsin Proteins 0.000 claims abstract description 35
- 239000012588 trypsin Substances 0.000 claims abstract description 35
- 238000001035 drying Methods 0.000 claims abstract description 17
- 238000011084 recovery Methods 0.000 claims abstract description 17
- 238000004140 cleaning Methods 0.000 claims abstract description 16
- 239000003513 alkali Substances 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 14
- 230000004151 fermentation Effects 0.000 claims abstract description 14
- 239000002253 acid Substances 0.000 claims abstract description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 91
- 239000000243 solution Substances 0.000 claims description 49
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 42
- 239000012535 impurity Substances 0.000 claims description 27
- 230000029087 digestion Effects 0.000 claims description 21
- 239000003929 acidic solution Substances 0.000 claims description 16
- 238000007598 dipping method Methods 0.000 claims description 13
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 11
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 11
- 239000012498 ultrapure water Substances 0.000 claims description 11
- 238000012545 processing Methods 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 2
- 238000002791 soaking Methods 0.000 abstract description 12
- 230000007613 environmental effect Effects 0.000 abstract 1
- 230000008092 positive effect Effects 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 238000005406 washing Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 6
- 239000000969 carrier Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000007654 immersion Methods 0.000 description 5
- 239000002699 waste material Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 2
- 230000006353 environmental stress Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000036284 oxygen consumption Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- 229920004935 Trevira® Polymers 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 208000032625 disorder of ear Diseases 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for recovering flaky carrier. The method is characterized by comprising the following steps of: (1) soaking the flaky carrier, which is taken out after cell fermentation is finished, with warm pure water and then cleaning; (2) soaking with alkali liquor, to be specific, soaking the flaky carrier treated by the step (1) in the alkali liquor and then cleaning; (3) digesting with trypsin solution, to be specific, soaking and digesting the flaky carrier treated by the step (2) in the trypsin solution and then cleaning; (4) soaking with acid liquor, to be specific, soaking the flaky carrier treated by the step (3) in acid solution to restore the functionality of the flaky carrier; and (5) drying: drying the flaky carrier treated by the step (4), namely removing moisture to dry the flaky carrier. The method is simple and feasible, and low in recovery cost and has a positive effect on environmental protection.
Description
Technical field
The present invention relates to a kind of method that reclaims chip carrier.
Background technology
Chip carrier; For example; The Fibra disk carrier that U.S. NBS company produces; The scraps of paper carrier that Taiwan match space tidal type reactor drum is used; The scraps of paper carrier that Hangzhou An Pu company torrent formula reactor drum is used,
Cephodisk carrier that the triumphant auspicious biotechnology of Binzhou, Shandong Province Bel ltd produces etc.Mostly chip carrier is disk shape, little rhomboid, " N " shape etc.; Adopt filamentary materials such as trevira, nanometer synthon to process; Its inside has fenestral fabric; These fenestral fabrics constitute the three-dimensional porous crack of wetting ability of chip carrier, can provide very high specific surface area (like 1gFibra disk carrier 1200cm can be provided for the cell growth
2The growth area), strengthen the attaching ability of cell, promote the cell rapid amplifying.Just because of this, carry out in the technology of large scale culturing zooblast at cell reactor, the application of chip carrier is very general.
In the mass cell fermenting process, the usage quantity of chip carrier can increase along with the increase of cell reactor scale, thereby in the cell reactor fermenting process, has occupied main cost.And chip carrier is a medical disposable material, and price is very expensive again, causes the cost of cell reactor fermentation very high; For example; The basket reactor drum of U.S. NBS company, the 5L reactor drum need add chip carrier 150g, and the 40L reactor drum need add chip carrier 1200g; Usage quantity is very big, and its market value is about 50 ~ 60 yuans/g.Chip carrier has occupied main cost as a kind of medical disposable material of costliness in this kind cell reactor fermenting process, thereby the production cost of cell fermentation is higher.Moreover,, be prone to environment is polluted, increase environmental stress if a large amount of chip carriers is arbitrarily abandoned stacking.
And chip carrier is after using, and cell debris and albumen impurity remain in chip carrier tridimensional network inside, are attached on the fibrous bundle; Be difficult to separate, thereby be difficult to clean up, if without exercise due diligence with its recycle; Very easily new cell growth is caused disadvantageous effect, cause growth conditions not good, in addition dead; And then the output and the quality of reduction title product, so rare people carries out the secondary use to chip carrier in the present industry.
Summary of the invention
For overcoming the technical problem that exists in the prior art, the object of the invention provides a kind of method that reclaims chip carrier, and this method is simple, and cost recovery is cheap, and environment protection is had active effect.
The objective of the invention is to realize through following technical scheme: a kind of method that reclaims chip carrier, it may further comprise the steps:
(1) pure water cleans: soak cell fermentation with warm pure water and finish the chip carrier that the back is taken out, clean then, detect by an unaided eye to colourless and inclusion-free until the pure water that cleans chip carrier, drain;
(2) dipping by lye: the chip carrier after step (1) is handled places alkali lye to soak, and cleans the water of pouring out after the extremely cleaning with pure water then and does not have the visible impurity of naked eyes, drains;
(3) trypsin solution digestion: the chip carrier after step (2) processing places trypsin solution to soak digestion, and then with the visible impurity of no naked eyes in the water of pouring out after pure water cleaning to the cleaning, drains;
(4) acid soak: it is functional to recover it that the chip carrier after step (3) is handled places acidic solution to soak, cleans up with ultrapure water then, and be neutral to pH value, the visible impurity of no naked eyes drains in the water of pouring out after the cleaning;
(5) drying: the chip carrier to after step (4) processing carries out drying treatment, removes moisture and makes it dry.
The present invention removes with warm pure water earlier and sticks to most cell debris and water-soluble albumen impurity on the chip carrier, because the relative cold water of warm water washes down cell debris and albumen impurity more easily from carrier; Use dipping by lye then, make residual protein denaturation, dissolving; And remaining small portion cell debris and albumen impurity that alkali lye is not removed are removed through tryptic digestion.Then, it is functional to recover it to soak said flaky carrier material with acidic solution again, is beneficial to cell when it is reused and attaches, and acid soak also has the effect of removing impurity in the chip carrier once more in addition, and is with as far as possible that the Impurity removal in the carrier is clean.
The embodiment that the present invention recommends is: the soak time of chip carrier in hot pure water can be 10 ~ 20 minutes in the described step (1); Described cleaning can be adopted the mode of softly washing by rubbing with the hands repeatedly, and detecting by an unaided eye until the pure water that soaks chip carrier is colourless and inclusion-free.
It is good that the middle warm pure water that soaks chip carrier of step according to the invention (1) should not have all chip carriers; Said warm pure water preferably adopts 40 ~ 60 ℃ of pure water to get final product; The pure water of this TR not only washes down cell debris and albumen impurity easily from carrier; And temperature is gentle moderate inviolent, is convenient to operator's manual processing.
Should not have all chip carriers be good to alkali lye when step according to the invention (2) was soaked.Preferably, described alkali lye and chip carrier ratio are 10 ~ 20ml/g, both can soak carrier fully, also can not cause the waste of alkali lye.Because concentration of lye is low excessively; Immersion does not reach the purpose of removing impurity in the carrier, and excessive concentration can cause unnecessary destruction to the internal structure of carrier; Therefore; It is analytical pure sodium hydroxide (NaOH) solution or analytical pure Pottasium Hydroxide (KOH) solution of 0.1 ~ 0.5mol/L that described alkali lye adopts preferred concentration, can at utmost remove in the carrier in the impurity reaching, and guarantees the integrity of carrier inside structure again.The present invention is analytical pure sodium hydroxide or the analytical pure Pottasium Hydroxide of 0.4 ~ 0.5mol/L more preferably.
Dipping by lye should make residual protein denaturation and dissolving in the chip carrier as far as possible in the step according to the invention (2); Preferred 0.5 ~ 4h of dipping by lye time among the present invention; Can make protein denaturation residual in the carrier in the scope at this moment, overlong time, the effect of dipping by lye has not had practical significance; And can prolong the treatment time, be unfavorable for improving processing efficiency.
The best pH value of trypsin solution digestion is between 7 to 8; This moment, eupepsy was stronger; Pretend and be a kind of preferred implementation of the present invention; In the said step (2), can the chip carrier after handling through alkali lye be cleaned to being weakly alkaline with clear water, can be beneficial to tryptic digestion in the step (3) like this.
The consumption of the trypsin solution of step according to the invention (3) should be good not have all chip carriers; Ratio between said trypsin solution and chip carrier is preferably in 10 ~ 20ml/g scope; Chip carrier was fully immersed in the trypsin solution; And can not cause the waste of trypsin solution, help practicing thrift the carrier recovery cost.
The trypsin solution immersion should not be removed alkali lye as far as possible in the step according to the invention (3) remaining small portion cell debris and albumen impurity digestion are removed.And the preferred 0.5 ~ 2h of trypsin solution soak time described in the present invention can fully digest the residual protein impurity that is attached in the carrier at this time range endotrypsin.
The concentration of the trypsin solution of step according to the invention (3) should be being entirely cell debris in the chip carrier and the digestion of albumen impurity good.The mass and size concentration of trypsin solution of the present invention preferably can reach the proteic effect of digestion impurity in 0.025% ~ 0.25% scope, do not cause tryptic waste again, and its concrete prescription is: 0.025 ~ 0.25g trypsinase, 100ml PBS solution.
The mass and size concentration of the trypsin solution of step according to the invention (3) more preferably in 0.15% ~ 0.25% scope, can reach the proteic best effect of digestion impurity, does not cause tryptic waste more than needed simultaneously.And the best digestion condition of trypsin solution is 37 ~ 38 ℃, thereby the digestion temperature in the said step (3) can be controlled in 37 ~ 38 ℃ of scopes.
The consumption of the acidic solution of step according to the invention (4) should be good not have all chip carriers; Preferably; Ratio between described acidic solution and chip carrier is in 10 ~ 20ml/g scope; Chip carrier is fully immersed in the acidic solution, and can not causes the waste of acidic solution, help practicing thrift the carrier recovery cost.
Acidic solution concentration is low excessively; The functional restoration that is unfavorable for chip carrier, excessive concentration then have the opposite effect to the functional restoration of chip carrier, therefore; The PH scope of the acidic solution of said step (4) is preferably 0 ~ 5; Can adopt 0.1 ~ 0.5mol/L hydrochloric acid soln, the hydrochloric acid soln of preferred 0.3 ~ 0.4mol/L adopts the acidic solution of above-mentioned concentration also can play the effect of removing once more to the minute quantity albumen impurity that remains in the chip carrier through step (2) and (3) processing continued in addition.
It is functional that the middle acidic solution immersion of step according to the invention (4) should fully recover chip carrier; Soak time is too short, and the functional restoration of carrier is incomplete, and overlong time increases the time of reclaiming; Therefore, acidic solution soak time 0.5 ~ 1h preferably in the said step (4).
The drying temperature of step according to the invention (5) is unsuitable too high, should be controlled in the scope of the material of can dry chip carrier not destroying chip carrier again and structure, avoids the material of chip carrier and structure to destroy because of dry high temperature.Drying temperature of the present invention preferably is controlled in 40 ~ 50 ℃ of scopes, and time of drying is preferably in 8 ~ 10 hours scopes.
Compared with prior art, the present invention has following beneficial effect:
1. method provided by the invention is simple; Cost recovery is cheap, and in the process of handling, the concentration and the drying temperature of basic soln, acidic solution are moderate; Be difficult for the material and the structure of chip carrier are damaged; Can make the recovery of chip carrier reach 100%, can reuse more than 4 to 5 times, more than 90% of effect before the application efficiency after the recovery can reach and reclaim.
Before the chip carrier that the present invention reclaims is being reused preferably again with the ultrapure water enchroachment (invasion) bubble of more cell cultures level more than 1 hour, use if mix with new chip carrier, effect is ideal more.
3. the chip carrier that method of the present invention reclaims can second stage employ; Not only reduced the production cost of cell fermentation; And reduced the accumulation of the chip carrier that abandons, reduce environmental pollution and alleviate environmental stress, so the present invention just there is active effect to environment protection.
Embodiment
Embodiment one:Reclaim the NBS Fibra disk of company carrier
(1) pure water cleans: after last consignment of time cell fermentation finishes; From cell reactor, take out chip carrier, soak 20 minutes with 40 ℃ warm pure water after, the soft repeatedly chip carrier of washing by rubbing with the hands; Water is constantly changed in the centre; Pure water until soaking chip carrier detects by an unaided eye to colourless, can't see impurity, drains with the leakage basin.
(2) dipping by lye: with NaOH (analytical pure) solution of pure water and analytical pure NaOH preparation 0.5mol/L; Will through the chip carrier after step (1) is handled and drained in proportion the ratio of 10ml/g immerse in this NaOH (analytical pure) solution; Soak and clean repeatedly with pure water after 1 hour; Be determined as till the weakly alkaline with the PH test paper, drain with the leakage basin.
(3) trysinization: use 0.25g trypsinase; The 100mlPBS compound concentration is 0.25% trypsin solution; Will through the chip carrier after step (2) are handled and drained in proportion the ratio of 10ml/g immerse in the trypsin solution 37 ~ 38 ℃ down digestion clean up repeatedly with pure water after 2 hours, drain with the leakage basin.
(4) acid soak: with pure water and concentrated hydrochloric acid preparation 0.5mol/L hydrochloric acid soln; Will through the chip carrier after step (3) are handled and drained in proportion the ratio of 10ml/g immerse in this hydrochloric acid soln; Soak and clean repeatedly with pure water after 0.5 hour, with the PH test paper measure to pH value near neutrality, clean repeatedly 3 to 6 times with ultrapure water again; Inclusion-free in the water of pouring out after the cleaning drains with the leakage basin.
(5) oven dry: put into electric oven 40 ℃ dry 10 hours down, remove the moisture on the chip carrier, make it drying.
The chip carrier that reclaims is steeped more than 1 hour at the ultrapure water enchroachment (invasion) with more cell cultures level, carry out cell fermentation by following method then, test cell consumes sugared amount, oxygen-consumption, viral Yield, virus titer parameter then, and the result is referring to table 1.
New NBS company's Fibra disk chip carrier and present embodiment recovery carrier are once carried out the parameter comparison experiment, and wherein reactor used model is NBS 510 type 40L reactor drums, adds carrier 12 00g, and cell strain is the PK-15 cell, and the inoculation total amount is 5*10
9Individual, connect malicious pig annulus 2 C-type virus Cs (PCV2), it is identical to meet malicious MOI.
Embodiment two: reclaim An Pu company scraps of paper carrier
(1) pure water cleans: after cell fermentation finishes; From cell reactor, take out scraps of paper carrier, with 60 ℃ warm pure water immersions after 10 minutes, the soft repeatedly chip carrier of washing by rubbing with the hands; Water is constantly changed in the centre; Pure water until soaking chip carrier detects by an unaided eye to colourless, can't see impurity, drains with the leakage basin.
(2) dipping by lye: with NaOH (analytical pure) solution of pure water and analytical pure NaOH preparation 0.3mol/L; Will through the chip carrier after step (1) is handled and drained in proportion the ratio of 15ml/g immerse in NaOH (analytical pure) solution; 0.5 clean repeatedly with pure water after hour; Be determined as till the weakly alkaline with the PH test paper, drain with the leakage basin.
(3) trypsin solution digestion: with the trypsin solution of 0.025g trypsinase and 100mlPBS compound concentration 0.025%; Will through the chip carrier after step (2) are handled and drained in proportion the ratio of 15ml/g immerse in the trypsin solution; Clean up repeatedly with pure water after 1 hour in digestion under 37 ~ 38 ℃, drain with the leakage basin.
(4) acid soak: with the hydrochloric acid soln of pure water and concentrated hydrochloric acid preparation 0.2mol/L; Will through the chip carrier after step (3) are handled and drained in proportion the ratio of 15ml/g immerse in the hydrochloric acid soln; Soak and clean repeatedly with pure water after 1 hour; With the PH test paper measure to pH value near neutrality, clean repeatedly 3 to 6 times with ultrapure water again, drain with the leakage basin.
(5) oven dry: put into electric oven 50 ℃ dry 8 hours down, remove the moisture on the chip carrier, make it drying.
Embodiment three: reclaimThe Cephodisk carrier
(1) pure water cleans: after last consignment of time cell fermentation finishes; From cell reactor, take out scraps of paper carrier, with 50 ℃ warm pure water immersions after 20 minutes, the soft repeatedly chip carrier of washing by rubbing with the hands; Water is constantly changed in the centre; Pure water until soaking chip carrier detects by an unaided eye to colourless, can't see impurity, drains with the leakage basin.
(2) dipping by lye: with NaOH (analytical pure) solution of pure water and analytical pure NaOH preparation 0.3mol/L; Will through the chip carrier after step (1) is handled and drained in proportion the ratio of 20ml/g immersed NaOH (analytical pure) solution 1 hour; Clean repeatedly with pure water afterwards; Be determined as till the weakly alkaline with the PH test paper, drain with the leakage basin.
(3) trypsin solution digestion: with the trypsin solution of 0.25g trypsinase and 100mlPBS preparation 0.25%; Will through the chip carrier after step (2) are handled and drained in proportion the ratio of 20ml/g immerse in the trypsin solution; Clean up repeatedly with pure water after 2 hours in digestion under 37 ~ 38 ℃, drain with the leakage basin.
(4) acid soak: with pure water and with the hydrochloric acid soln of concentrated hydrochloric acid preparation 0.4mol/L; Will through the chip carrier after step (3) are handled and drained in proportion the ratio of 20ml/g immerse in this hydrochloric acid soln; Soak and clean repeatedly with pure water after 0.5 hour; With the PH test paper measure to pH value near neutrality, clean repeatedly 3 to 6 times with ultrapure water again, drain with the leakage basin.
(5) oven dry: put into electric oven 45 ℃ dry 9 hours down, remove the moisture on the chip carrier, make it drying.
The chip carrier that reclaims is steeped more than 1 hour at the ultrapure water enchroachment (invasion) with more cell cultures level, carry out cell fermentation then, test cell consumes sugared amount, oxygen-consumption, viral Yield, virus titer parameter then, and the result is referring to table 2.
New sheet support C ephodisk is carried out the parameter comparison experiment with the carrier that reclaims once, and wherein reactor used model is NBS 510 type 40L reactor drums, adds carrier 1100g, and cell strain is the Marc-145 cell, and the inoculation total amount is 4*10
9Individual, connect malicious pig blue-ear disease poison (PRRSV), it is identical to meet malicious MOI.
Embodiment four:
(1) pure water cleans: after last consignment of time cell fermentation finishes; From cell reactor, take out chip carrier, soak 15 minutes with 45 ℃ warm pure water after, the soft repeatedly chip carrier of washing by rubbing with the hands; Water is constantly changed in the centre; Pure water until soaking chip carrier detects by an unaided eye to colourless, can't see impurity, drains with the leakage basin.
(2) dipping by lye: with KOH (analytical pure) solution of pure water and analytical pure KOH preparation 0.45mol/L; Will through the chip carrier after step (1) is handled and drained in proportion the ratio of 10ml/g immersed this KOH (analytical pure) solution 4 hours; Clean repeatedly with pure water afterwards; Be determined as till the weakly alkaline with the PH test paper, drain with the leakage basin.
(3) trysinization: use 0.15g trypsinase and 100mlPBS compound concentration are 0.15% trypsin solution; Will through the chip carrier after step (2) are handled and drained in proportion the ratio of 12ml/g immerse trypsin solution; Clean up repeatedly with pure water after 1.5 hours in digestion under 37 ~ 38 ℃, drain with the leakage basin.
(4) acid soak: with pure water and concentrated hydrochloric acid preparation 0.35mol/L hydrochloric acid soln; Will through the chip carrier after step (3) are handled and drained in proportion the ratio of 12ml/g immerse in this hydrochloric acid soln; Clean repeatedly with pure water after 45 minutes, with the PH test paper measure to pH value near neutrality, clean repeatedly 3 to 6 times with ultrapure water again; Inclusion-free in the water of pouring out after the cleaning drains with the leakage basin.
(5) oven dry: put into electric oven 45 ℃ dry 10 hours down, remove the moisture on the chip carrier, make it drying and get final product.
Embodiment five:
(1) pure water cleans: after last consignment of time cell fermentation finishes; From cell reactor, take out chip carrier, soak 15 minutes with 45 ℃ warm pure water after, the soft repeatedly chip carrier of washing by rubbing with the hands; Water is constantly changed in the centre; Pure water until soaking chip carrier detects by an unaided eye to colourless, can't see impurity, drains with the leakage basin.
(2) dipping by lye: with KOH (analytical pure) solution of pure water and analytical pure KOH preparation 0.45mol/L; Will through the chip carrier after step (1) is handled and drained in proportion the ratio of 18ml/g immersed this KOH (analytical pure) solution 4 hours; Clean repeatedly with pure water afterwards; Be determined as till the weakly alkaline with the PH test paper, drain with the leakage basin.
(3) trysinization: use 0.2g trypsinase and 100mlPBS compound concentration are 0.2% trypsin solution; Will through the chip carrier after step (2) are handled and drained in proportion the ratio of 18ml/g immerse trypsin solution; Clean up repeatedly with pure water after 1.5 hours in digestion under 37 ~ 38 ℃, drain with the leakage basin.
(4) acid soak: with pure water and concentrated hydrochloric acid preparation 0.3mol/L hydrochloric acid soln; Will through the chip carrier after step (3) are handled and drained in proportion the ratio of 12ml/g immerse in this hydrochloric acid soln; Clean repeatedly with pure water after 45 minutes, with the PH test paper measure to pH value near neutrality, clean repeatedly 3 to 6 times with ultrapure water again; Inclusion-free in the water of pouring out after the cleaning drains with the leakage basin.
(5) oven dry: put into electric oven 40 ℃ dry 10 hours down, remove the moisture on the chip carrier, make it drying and get final product.
Embodiment six:
(1) pure water cleans: after last consignment of time cell fermentation finishes; From cell reactor, take out chip carrier, soak 10 minutes with 55 ℃ warm pure water after, the soft repeatedly chip carrier of washing by rubbing with the hands; Water is constantly changed in the centre; Pure water until soaking chip carrier detects by an unaided eye to colourless, can't see impurity, drains with the leakage basin.
(2) dipping by lye: with KOH (analytical pure) solution of pure water and analytical pure KOH preparation 0.1mol/L; Will through the chip carrier after step (1) is handled and drained in proportion the ratio of 20ml/g immersed this KOH (analytical pure) solution 3 hours; Clean repeatedly with pure water afterwards; Be determined as till the weakly alkaline with the PH test paper, drain with the leakage basin.
(3) trysinization: use 0.1g trypsinase and 100mlPBS compound concentration are 0.2% trypsin solution; Will through the chip carrier after step (2) are handled and drained in proportion the ratio of 20ml/g immerse trypsin solution; Clean up repeatedly with pure water after 0.5 hour in digestion under 37 ~ 38 ℃, drain with the leakage basin.
(4) acid soak: with pure water and concentrated hydrochloric acid preparation 0.1mol/L hydrochloric acid soln; Will through the chip carrier after step (3) are handled and drained in proportion the ratio of 20ml/g immerse in this hydrochloric acid soln; Clean repeatedly with pure water after 45 minutes, with the PH test paper measure to pH value near neutrality, clean repeatedly 3 to 6 times with ultrapure water again; Inclusion-free in the water of pouring out after the cleaning drains with the leakage basin.
(5) oven dry: put into electric oven 60 ℃ of oven dry down, remove the moisture on the chip carrier.
The present invention can summarize with other the specific form without prejudice to spirit of the present invention or principal character.The above embodiment of the present invention can only think all that to explanation of the present invention rather than restriction every foundation essence technology of the present invention all belongs in the scope of technical scheme of the present invention any trickle modification, equivalent variations and modification that above embodiment did.
Claims (10)
1. a method that reclaims chip carrier is characterized in that, may further comprise the steps:
(1) pure water cleans: soak cell fermentation with warm pure water and finish the chip carrier that the back is taken out, clean then, detect by an unaided eye to colourless and inclusion-free until the pure water that cleans chip carrier, drain;
(2) dipping by lye: the chip carrier after step (1) is handled places alkali lye to soak, and cleans the water of pouring out after the extremely cleaning with pure water then and does not have the visible impurity of naked eyes, drains;
(3) trypsin solution digestion: the chip carrier after step (2) processing places trypsin solution to soak digestion, and then with the visible impurity of no naked eyes in the water of pouring out after pure water cleaning to the cleaning, drains;
(4) acid soak: the chip carrier after step (3) is handled places acidic solution to soak to recover its function, cleans up with ultrapure water then, and be neutral to pH value, the visible impurity of no naked eyes drains in the water of pouring out after the cleaning;
(5) drying: the chip carrier to after step (4) processing carries out drying treatment, removes moisture and makes it dry.
2. the method for recovery chip carrier according to claim 1 is characterized in that, in the said step (2), the chip carrier after handling through alkali lye is cleaned to being weakly alkaline with pure water.
3. the method for recovery chip carrier according to claim 1 and 2 is characterized in that, described alkali lye of said step (2) and chip carrier ratio are 10 ~ 20ml/g; It is analytical pure sodium hydroxide solution or the analytical pure potassium hydroxide solution of 0.1 ~ 0.5mol/L that described alkali lye adopts concentration; The said dipping by lye time is 0.5 ~ 4h.
4. the method for recovery chip carrier according to claim 3 is characterized in that, the ratio described in the said step (3) between trypsin solution and chip carrier is in 10 ~ 20ml/g scope.
5. the method for recovery chip carrier according to claim 4 is characterized in that, the trypsin solution soak time is 0.5 ~ 2h in the said step (3); The mass and size concentration of described trypsin solution is in 0.025% ~ 0.25% scope.
6. the method for recovery chip carrier according to claim 5 is characterized in that, the mass and size concentration of the trypsin solution of said step (3) is in 0.15% ~ 0.25% scope.
7. the method for recovery chip carrier according to claim 6 is characterized in that, the acidic solution of said step (4) and the ratio between chip carrier are in 10 ~ 20ml/g scope.
8. the method for recovery chip carrier according to claim 7 is characterized in that, described acidic solution adopts 0.1 ~ 0.5mol/L hydrochloric acid soln.
9. the method for recovery chip carrier according to claim 8 is characterized in that, the acidic solution soak time is 0.5 ~ 1h in the said step (4).
10. the method for recovery chip carrier according to claim 9 is characterized in that, the drying temperature of said step (5) is in 40 ~ 50 ℃ of scopes, and time of drying is in 8 ~ 10 hours scopes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210038683.0A CN102586107B (en) | 2012-02-21 | 2012-02-21 | Method for recovering flaky carrier |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101576653A (en) * | 2009-06-22 | 2009-11-11 | 中国医科大学附属第一医院 | Method for washing and recycling membranate glass slide for microdissection |
| CN102133504A (en) * | 2010-01-25 | 2011-07-27 | 滕国新 | Filter membrane cleaning agent and cleaning technology |
| CN102146356A (en) * | 2010-06-24 | 2011-08-10 | 辽宁成大生物股份有限公司 | Method for regenerating microcarrier |
| CN102174497A (en) * | 2010-12-30 | 2011-09-07 | 北京民海生物科技有限公司 | Recycling method of microcarrier |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101576653A (en) * | 2009-06-22 | 2009-11-11 | 中国医科大学附属第一医院 | Method for washing and recycling membranate glass slide for microdissection |
| CN102133504A (en) * | 2010-01-25 | 2011-07-27 | 滕国新 | Filter membrane cleaning agent and cleaning technology |
| CN102146356A (en) * | 2010-06-24 | 2011-08-10 | 辽宁成大生物股份有限公司 | Method for regenerating microcarrier |
| CN102174497A (en) * | 2010-12-30 | 2011-09-07 | 北京民海生物科技有限公司 | Recycling method of microcarrier |
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