CN1333337A - Batch production for age synchronization cell - Google Patents
Batch production for age synchronization cell Download PDFInfo
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- CN1333337A CN1333337A CN 01103282 CN01103282A CN1333337A CN 1333337 A CN1333337 A CN 1333337A CN 01103282 CN01103282 CN 01103282 CN 01103282 A CN01103282 A CN 01103282A CN 1333337 A CN1333337 A CN 1333337A
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Abstract
The present invention relates to a method for producing cell population with identical age, and is characterized by that the cell with some defined age can be harvested on one surface, and in the next continuous culture process the harvested cells can be remained on the above-mentioned surface, and their filial generation can be separated so as to prevent them from mixing with their posterity. In proper time, from said surface the sample can be taken, so that the cell population with required age can be obtained, can be used for inspection, test and other different applications.
Description
The present invention sets forth a kind of method with the cell colony of collecting the age unanimity and keep the synchronization at this cell colony age.
Since nineteen fifty, existing the whole bag of tricks is used to obtain the cell colony of " synchronization " and (sees " synchronization of cell fission and growth " that E.Zeuthen work Interscience press published in New York in 1964, C.E.Helmstetter was published in " Enzymology method " (Meth.Enzymol.) paper of the 1st volume 327-363 page or leaf in 1969, and W.Krek and J.A.DeCaprio1995 are published in " Enzymology method " (Meth.Enzymol.) paper of the 254th volume 114-124 page or leaf).These methods can be classified as to be selected synchronization and induces synchronization two big classes.
In selecting synchronization, a certain physicochemical characteristics is used as the cellular segregation based on " cell cycle " (essence is " germiparity cycle ") in the cell separation stage.These physicochemical characteristicses that are utilized can be the inherent feature of cell own such as cell size and cell density, also can be that the people is that the feature of giving is as molecule that is incorporated into the tape label in the biomass or the molecule that is affixed on the tape label of cell surface.
In inducing synchronization, the cell that is in different developmental phases is stopped at or is induced to a certain specified etap: in due course, these cells are allowed to enter next etap at one time and begin synchronization.The material that prevention commonly used or inducing cell are grown normally those abilities gives or promotes the material of " cell cycle " (essence is " germiparity cycle ").
The cell synchronization device of " filial generation machine " (Baby Machine) was developed and (saw that C.E.Helmstetter and D.J.Cummings1963 are published in " American Academy of Sciences's circular " (Proc.Natl.Acad.Sci.USA) paper of 5Q volume 767-774 page or leaf a kind of being called as, C.E.Helmstetter1991 is published in the paper of " neontology " (New Biol.) the 3rd volume 1089-1096 page or leaf and C.E.Helmstetter etc. and was published in " bacteriology magazine " (J.Bacteriol.) paper of the 174th volume 3445-3449 page or leaf in 1992).By this approach, discharge the daughter cell that gets off and be received in incubation growth together from being combined in cell on the filter paper.Though wish so can obtain the synchronization of the cell cycle of culture of continuous cultivation midium or long term, can in fact this hope never was implemented.Comprise when this method is used to intestinal bacteria and bacto yeast synchronized (seeing that C.E.Helmstetter1991 is published in the paper of " neontology " (New Biol.) the 3rd volume 1089-1096 page or leaf and C.E.Helmstetter etc. and was published in " bacteriology magazine " (J.Bacteriol.) paper of the 174th volume 3445-3449 page or leaf in 1992).
A kind of dull and stereotyped release tech has been used to the synchronization (seeing that S.T.Degnen and A.Newton1972 are published in " molecular biology magazine " (J.Mol.Biol.) paper of the 64th volume 671-680 page or leaf) to handle bar mattress.Handle bar mattress is called as asymmetric thin mattress because of the group born of the same parents that split into a swarm cell and a band handle bar.Because the vertical sucker of handle bar has viscosity, the cell of band handle bar can be attached to table molecular biology magazine " (J.Mol.Biol.) paper of the 64th volume 671-680 page or leaf).Handle bar mattress is called as asymmetric bacterium because of the group born of the same parents that split into a swarm cell and a band handle bar.Because the vertical sucker of handle bar has viscosity, the cell of band handle bar can be attached to the surface as on the culture dish flat board, and still is attached to the there in the fission process afterwards.In contrast, swarm cell has power owing to having an extreme flagellum.In case just separate and to swim the liquid from the handle rod that is attached to the surface.Therefore, the synchronization that handle bar mattress is carried out the cell age only need be collected the swarm cell that comes from the handle rod division of pasting in a short time and just can be reached.Yet a lot of researchs are proof repeatedly, and as synchronized swarm cell of these ages is continued to cultivate, the synchronization of cell cycle will be lost since second cell cycle.This is because though the handle rod can just carry out division next time after finishing first cell cycle, swarm cell just can enter the next cell cycle after must growing up to the handle rod earlier.Therefore, as reaching the successive cell cycle synchronization of handle bacillus, must repeat density centrifugation to separate this cell of two types.This consumption of centrifugal process repeatedly power is consuming time.Therefore seldom extend to second cell cycle of its vital process about the research of handle bacillus.
There is certain methods to be developed the old bacto yeast cell that germinates to obtain.Wherein a kind of method is to utilize the difference on existing size/density between bigger parent cell and the less sprout cell and to carry out the circular frequency of sucrose density gradient repeatedly centrifugal to separate big old cell and little young cell nineteen nineties such as (see be published in " old bio-science magazine " (J.Gerontol.Biol.Sci.) paper of the 45th volume B9-B17 page or leaf) N.K.Egilmez.Other method depends on the vitamin H cell of mark youth optionally, the combining of avidin on utilizing vitamin H and be enclosed in the magnetic grain after these cells elder and obtain to have biotin labeled old cell (seeing that T.J.Smeal etc. was published in " cell " (Cell) paper of the 84th volume 633-642 page or leaf in 1996).
As everyone knows, the method of all known cell synchronizations all can not be kept fissional being synchronized to more than several cell cycles, no matter processed cell is the protokaryon unicellular microorganism, eucaryon unicellular microorganism, or the histocyte of eucaryon multicellular organism (seeing that " synchronization of cell fission and growth " that E.Zeuthen work Interscience press published in New York in 1964 and C.E.Helmstetter etc. were published in " bacteriology magazine " (J.Bacteriol.) the 174th paper of rolling up the 3445-3449 page or leaf in 1992).It is a unsolved riddle all the time that yet synchronized cell loses synchronism in actually why can cultured continuously afterwards so soon.
Existing method for synchronizing either overt or covertly is all claimed so hypothesis, and promptly two cells that come by a cell fission are two sister cells of belonging to the same generation and have the identical age (seeing that outstanding Garland such as " physiology of bacterial cell " that work Sinauer Associates company such as F.C.Neidhardt published in masschusetts, u.s.a Sunderland city in nineteen ninety and B.Alberts press was in " molecular biology of cell " in the USA New York publication in 1989).Because this is spread wide but in fact also unconfirmed hypothesis, generally all think in case a cell colony is to obtain at same cell separation stage, in its later cultivation, will automatically give division stage (usually being takeed for the age) synchronized cell so.
Yet, the viewpoint of this doctrinal cell life and cell synchronization and the cell life of many forms real inconsistent and also be wrong (seeing that S.V.Liu2000 is published in the paper of " logic biology " (Logical Biology) the 2000th volume 5-16 page or leaf) logically.A new cell life modes proposes, a cell fission and in fact two cells coming should belong to two generations that pass on from one to another up and down and have the different ages (paper that S.V.Liu1999 is published in " Chinese science " (Science in China) the 42nd volume 644-654 page or leaf).If this new theory is correct, so just mean that the basic assumption that most of existing cell synchronization method relies on is untenable.
Conclusion is got up, and existing cell synchronization method has following one or more defectives:
(a) the initial cell colony that is used to begin cell synchronization often has the different ages.A lot of for a long time people think that being in same division (reproduction) stages of cell has the identical age.Yet this hypothesis does not reflect all reality, and is untenable in logic yet.For example, say that all are about to the splitted cell is age-grade cell, just as saying that all antenatal mothers have same age.This saying is also not exclusively true.Even show as the synchronization on the reproductive cycle because the women of different ages also might begin pregnancy at one time.
(b) even initiator cell group's division stage or age are synchronized, its cell that continues different generations in the culturing process still mixes.The hypothesis that parent cell is split into two sister cells has been violated biological fundamental principle, i.e. reproduction means goes down to posterity and the continuation of genetic material.Although the elimination of this false supposition is still needed further scientific research, point out the logic error of this debate and not so difficult with the contradiction of life principle of generality.For example, whoever can not believe that a women's federation just becomes one of her offspring after her fertility.Therefore, as in filial generation and female generation, not separated, even begin be the cell colony of age synchronization also can be when it continues to cultivate spontaneous generation up and down generation mixing and cause the unhomogeneity at age.The life of being familiar with the most with us is that the mankind are the example explanation, if allow the newborn infant still to stay together with their mother, whoever can not believe this colony age that still tool is identical and the synchronization at the age of maintenance.
(c) the existing method for synchronizing of inducing only can reach the cell fission synchronization in (reproduction) cycle, but this does not mean the synchronization at cell age.Theoretically, the material of influential cell cycle (germiparity cycle) should effect all be arranged to all cells, no matter their age is how.Because so just may make the cell of different ages be parked in or be induced into same stage reproductive cycle.From this point, synchronized cell colony might not be to go up synchronously at the cell age on the cell cycle.
(d) cumbersome and non-natural process is used to the synchronization at cell age.Up to the present, only there is the method for only a few can obtain the cell colony of age greater than the age homogenization truly in a germiparity cycle.These methods are used to obtain old bacto yeast cell (see nineteen ninety such as N.K.Egilmez be published in " old bio-science magazine " (J.Gerontol.Biol.Sci.) paper of the 45th volume B9-B17 page or leaf and T.J.Smeal etc. were published in " cell " (Cell) paper of the 84th volume 633-642 page or leaf in 1996).Yet these methods need cumbersome process and special material or equipment.For example, in order to collect old yeast cell and they and young sprout cell separate with the long-term cultivation of maintenance to initiator cell, need carry out repeatedly centrifugal.This centrifugal repeatedly trouble and consume manpower, and the cell non-natural physiological condition of experience in the reason process herein.The method that another one is used for collecting the yeast cell colony of given age needs earlier with biotin labeling filial generation yeast cell, collects institute's labeled cell (seeing that T.J.Smeal etc. was published in " cell " (Cell) paper of the 84th volume 633-642 page or leaf in 1996) from the beginning with the vitamin H specific combination with the avidin 9 that is coated in magnetic grain surface then behind these cells elder.This method changes cell characteristics artificially, and can only be used for the cell that those can be admixed to vitamin H cell surface.It is magnetic separator that this method also needs a special equipment.
Another technology that can obtain the given age cell with future is the wandering cells instrument, if cell can specify the age to be labeled and this tagged molecule can be followed the tracks of by the wandering cells instrument.Yet, if the process of collecting cell is dispersed in long time range, the difference on still can has age between the collected different cells.Therefore, perhaps the wandering cells instrument not too is suitable for being used for obtaining the cell of a large amount of age synchronizations.And this method is expensive certainly.
In essence, the method for all " once the step " synchronizations (i.e. expectation obtains the same division stage or age-grade initiator cell group can reach long-term cell synchronization) is the cell mass that can't produce real age synchronization in follow-up cultivation theoretically.Those " multistep " method for synchronizing often need centrifugation repeatedly again or need first mark to obtain the cell of mark then.These methods can be collected the old cell of real age homogeneous.But spend a lot of time, expense and manpower.The cell that obtains as this method often experiences factitious living condition.Perhaps, these artificial pressure can disturb the research to these cell physiological states.
The objective of the invention is to correct the basic mistake of above-mentioned traditional method and overcome the deficiency of existing modification method and create a kind of natural, easy, economical, easily capable, be suitable for real-time monitoring, meet the different scales needs, can obtain the method for the cell colony of real age synchronization.
The present invention is based on the theory of the new cell life that the inventor proposes, i.e. a cell fission and have parent between two cells coming and have the different ages.From this viewpoint, the inventor thinks must constantly separate the age synchronization of the cell colony that will realize real meaning parental cell with their daughter cell.Therefore the objective of the invention is to reach by following measure:
(a) collect initial cell colony in a short period of time to a surface,
(b) in the cell aging process, constantly remove their filial generation and make the surface only have the cell colony of initial collection,
(c) in the cell aging process, get every now and then as required part surface with results different ages synchronized cell or monitor
Observe.
The combination of above step makes the present invention all different with existing cell synchronization method.At first, initiator cell colony is captured to a surface to have the age homogeneity of High Level to guarantee initiator cell colony in a very short time.The second, captive cell when the cell that is retained only is initial, and their daughter cell is ceaselessly removed, to keep initiator cell colony purity in years.The 3rd, the quality of surfacing and quantity can suitably be selected as required flexibly.For example can use transparent surfacing to catch initiator cell colony monitors to make things convenient for the synchronized process of pair cell to carry out real-time continuous.Initiator cell colony is captured to separate respectively different surfaces so that repeatedly sample and can not influence whole cell synchronization process at the different cell age.
Compare with other modification method with ordinary method, the present invention has the following advantages:
The present invention not only obtains the initiator cell colony of height age homogenization, but also constantly keeps the purity at its age in follow-up culturing process.In the filial generation machine, the cell that is fixed to the surface is taken from the culture of logarithmic phase usually.Extensively distribute from the cell size, in fact this phase comprises the cell at various ages.In addition, compare with the length in germiparity cycle, cell also is oversize with the time that the surface contacts.It is bigger that this way of collecting initial cell mass in long-time scope causes institute to obtain intercellular age difference.Because these shortcomings, the cell that obtains does not divide in synchronized mode.If cell synchronization is since an age blended cell colony, it will be very difficult wanting to reach secular cell age synchronization, if not complete impossible words.The present invention has overcome these shortcomings, and by only catch newborn cell in the time of lacking very much, the present invention can guarantee initiator cell group high level of synchronizationization in years.Though the filial generation machine can obtain daughter cell with the beginning cell synchronization, it not at it after in this initiator cell group's the cultivation segregant for and parental cell.Therefore cause the mixing at cell age in the follow-up culture.The present invention overcomes this age blended shortcoming by the offspring who constantly removes them from initial captured cell.
The present invention uses more natural and simple method.Compare with those methods that are used to obtain old yeast cell, the present invention utilizes some cells can adhere to these characteristics of surface automatically, thereby needn't use any strength that adds to collect these microorganisms by force.In addition, the present invention also can adopt some attractive surfaces that the cell that can not adhere to the surface is naturally collected.Just can reach long-term synchronized method and compare by centrifugal repeatedly with some, the present invention represents a kind of by nature or reach very much the method for cell synchronization near the process of nature.
In addition, the present invention also has the following advantages:
(a) expense on equipment and the reagent is very low.
(b) can obtain synchronized cell apace.
(c) has the handiness of satisfying different needs.
(d) be convenient to synchronization process is monitored in real time.
(e) be convenient to obtain cell at different ages.
(f) be convenient to automatization.
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 symbolically shows the cell that obtains the age homogenization, and keeps the method for age purity in its cultured continuously process.
Obtaining of initiator cell colony to the age homogenization can be undertaken by different modes, and in a kind of recommended method, a large amount of unsynchronized cells are allowed to adhere to a surface.This surface that is stained with cell should be washed for several times and adhere to not firm cell to remove.Then the container that another contains new substratum is put into adherent cell direction up in this surface, so that the continued growth from the teeth outwards of the cell of secure adhesion.Moment before the reproduction time of estimating arrives is put into another surface from the very near but direct position contacting of adherent cell, and the storage period on this new surface should be very short, so that have minimum age difference between captured cell.
Another kind method is to use different materials that cell development is stopped at or is induced to a certain specified phases.Allow these cells to carry out reproduction then and in very short time, neonatal cell captured a new surface.
Also having a kind of method of collecting the cell colony of age homogenization, is to specify the cell at age to carry out mark, utilizes the method that can combine with these labeled cells to collect the cell that is labeled then.Preferably these marks only mix neonatal cell, this like cell can be at birth or birth moment soon be collected.For example, vitamin H can be used to the mark neonatal cell, these the mark neonatal cell can with the cellular segregation at other age.Its way is with being combined with vitamin H by the avidin on magnetic grain surface.
Keeping of pair cell age purity can utilize the method for different removal daughter cells to reach in follow-up culturing process.In a method of recommending, the liquid stream of a horizontal direction can be used to wash away the daughter cell that falls down from parent cell.Inject new substratum continuously to culturing room, and get rid of old substratum, just can create this liquid stream to take away the cell of disengaging.
Another kind method is that the surface that will contain adherent cell rises from liquid level every now and then and puts liquid into, creates a kind of disturbance like this to help the disengaging of daughter cell.Liquid nutrient medium is changed every now and then avoiding or at least greatly to reduce daughter cell and is adhered on the surface of containing initiator cell.
In order to reduce operational expense, the exhausted substratum can be by filtering to remove contained cell.This substratum that does not contain cell and conditioning again can be recycled once more.
It can be any material that the present invention listens the surfacing of use, as long as this material has enough adhesive powers can maintain these cells in the culturing process of growing to the interested cell of institute.These surfacings include but not limited to plastic and glass.The surface of these materials can be untreated, also can be with other mass treatment cross with the attraction that strengthens pair cell and the adhesion of pair cell.This material that is used for treat surface includes but not limited to that left-handed poly relies peace acid.
In order to illustrate how the present invention reaches the synchronization at cell age, Fig. 1 symbolically describes its total process.A culturing room 101 is filled by substratum 102.This substratum contains the not cell at synchronized various ages.Though the difference of these cells on the age is not determined, can find out from its different size and form, as cell 103 before dividing, grown cell 104 that has extended and newborn minicell 105.
In order to collect as the used parent cell of synchronization in the future, a surface dull and stereotyped 106 is placed in the surface of liquid culture.Because adhesive power or because passive some cells of attraction are adsorbed to surface treated, these cells are in different ages and different reproductive phase probably itself.The surface that is stained with cell need be with fresh substratum washing to remove the not firm cell of any adhesion.Whole then surface is placed in another new substratum with cell direction up.
Give birth to the neonatal cell that gets off in order to collect from the adhesion parent cell, a new surface dull and stereotyped 107 is put in the place of closing on adherent cell.Very short for some time is only placed at this in this new surface, and feasible like this obtaining has only very little age difference between the neonatal cell, shown in the cell 110 of homogeneous size and form.Then, this surperficial flat board that contains neonatal cell is placed in the new substratum of another new container, from then on begins the synchronization process at cell age.
In ensuing culturing process, above-mentioned neonatal cell elder gradually forms the hatching cell 111 of making a living, and final production filial generation 112.Purity for age of keeping initiator cell is necessary to prevent that neonatal cell from also adhering on the surface.For reaching this purpose, available liquid stream 113 is washed the filial generation that has broken away from off.
Along with the prolongation of incubation time, it is old and feeble that initiator cell will continue, shown in increasing as cell darkness in drawing by slash 111 to intersect 114, to deceiving 115 entirely.Yet owing to constantly remove their filial generation, the cell colony of initial collection remains the age purity of height.
To the collection of the cell of age synchronization, can carry out in the arbitrary required time of synchronization process.The mode of collecting also can be multiple, gets certain surface as extracting the part from a bigger surface or containing the matrix on a plurality of surfaces from one.These cells that are collected can be used to multiple uses such as morphologic observation, Physiology and biochemistry test, heredity and genome research.
Should be pointed out that being fit to cell of the present invention includes but not limited to unicellular organism such as bacterium and yeast, might be with the histocyte of this invention synchronization multicellular organism.Therefore, scope of the present invention should should not decided with its jural explanation with additional claim with given example.
Claims (10)
1. method that produces age synchronization cell colony in the long-term cultivation process comprises:
(a) a kind of means of initial cell colony of collecting the age homogeneous and
(b) a kind of same means qualitatively of initial cell colony's age that in continuing culturing process, keep.
2. the claim 1 indication means of obtaining the initial cell colony of age homogeneous comprise cell fixation to the means of a certain designated surface with only collect the neonatal cell that the do not attach means to a certain new surface.
3. claim 2 indication surfaces can be selected from following one group of material comprising glass, plastics, fiber, paper and metal.
4. claim 2 indication surfaces can scribble the material such as the left-handed poly-lysine of energy adherent cell and attraction cell.
5. the means of the initial cell of claim 1 indication results age homogenization are included in designated cell age indicator cell and collect the means that are labeled cell specifically.
6. the means of claim 5 indication labeled cells comprise that the following one group of material of use is comprising vitamin H.
7. the method for claim 5 indication specificitys collection labeled cell comprises that the following method of use is comprising the part combination.
8. the claim 1 indication means of keeping initial cell colony age homogeneity comprise that following mode creates liquid-flow to separate its offspring's cell from adherent initiator cell:
(a) liquid nutrient medium is flow through the surface of containing above-mentioned cell with horizontal direction,
(b) liquid nutrient medium is flow through the surface of containing above-mentioned cell with vertical direction,
(c) surface that will contain above-mentioned initial cell emersion frequently, immerse again then liquid nutrient medium and
(d) import discontinuously and export discontinuously to culturing room and be used to cultivate the liquid nutrient medium of initial cell that sticks to the surface from culturing room.
9. claim 1 described cell includes, without being limited to the histocyte of various types of microorganism cellss and different sources.
10. claim 1 described cell age homogeneous and cell age synchronization are meant the cell age homogeneous and the cell age synchronization of the relative level of the certain limit that general Institute of Science is accepted.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102719480A (en) * | 2012-07-08 | 2012-10-10 | 大连医科大学 | Pre-processing method of cell culture plate for suspension cell liposome transfection |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719480A (en) * | 2012-07-08 | 2012-10-10 | 大连医科大学 | Pre-processing method of cell culture plate for suspension cell liposome transfection |
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