WO2018095323A1 - 活细胞传感仪在植物细胞低温保藏中的应用 - Google Patents

活细胞传感仪在植物细胞低温保藏中的应用 Download PDF

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WO2018095323A1
WO2018095323A1 PCT/CN2017/112299 CN2017112299W WO2018095323A1 WO 2018095323 A1 WO2018095323 A1 WO 2018095323A1 CN 2017112299 W CN2017112299 W CN 2017112299W WO 2018095323 A1 WO2018095323 A1 WO 2018095323A1
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cell
cells
cryopreservation
sucrose
cell viability
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郭美锦
李佳瑞
王泽建
陈雨霞
庄英萍
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East China University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G01N27/22—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating capacitance

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  • the invention belongs to the field of cell detection, and more particularly, the invention relates to the application of a living cell sensor in cryopreservation of plant cells.
  • the preservation of existing plant cell lines is mainly carried out in the following two ways.
  • the first is subculture in growth medium, and the other is cryopreservation of cells.
  • Preserving cells by long-term passage is time consuming, labor intensive, costly, and has the disadvantage of disappearing plant cell characteristics.
  • the cells When the cells are under ultra-low temperature conditions, they will stop a series of internal metabolic activities and maintain the characteristics of the cells themselves, greatly reducing the risk of disappearing cell characteristics.
  • the method of cryopreservation plays a decisive role in the success of the cells. Because plant cells contain a lot of water, it is difficult to cryopreservation. Therefore, it is necessary to select a suitable cryoprotectant and optimize a suitable cryopreservation method to most effectively control the intracellular water content and the damage to cells during the freezing process, so as to obtain higher cell activity after cryopreservation of the cells.
  • the living cell sensor uses a capacitive method to measure the concentration of living cells in a cell solution.
  • the alternating electric field present in the cell solution causes the ions therein to migrate, while the living cells have a complete insulating cell membrane in which the ions are bound to cause polarization of the cell membrane, which makes the cells become tiny capacitors.
  • the measured capacitance value has a good linear relationship with the amount of cells in the solution.
  • living cell sensors are often used in the fermentation process to determine the amount of cells in the fermentation broth, which has not been applied to cell cryopreservation.
  • a method of increasing cell viability of cryopreserved cells comprising:
  • DMSO 8 to 12% (w/v); more preferably 9 to 11% (w/v);
  • Sucrose 8 to 12% (w/v); more preferably 9 to 11% (w/v).
  • the step (1) of the method further comprises: measuring and recording the capacitance value A 1 on the pretreated cells;
  • step (2) after cryopreservation, the method further comprises: performing cell resuscitation on the plant cell, and measuring and recording the capacitance value A 2 by using a living cell sensor;
  • the cell base capacitance value is A 3 ;
  • the cell viability preservation rate is obtained by the formula (I):
  • the cell viability of the plant cells was evaluated based on the obtained cell viability preservation rate.
  • the plant cell is a momordica plant cell; preferably a mangosteen callus cell.
  • the sucrose is present in a liquid medium, and the plant cells in the logarithmic growth phase are added to the liquid medium for pretreatment.
  • the concentration of the sucrose is 0.18 to 0.22 M; more preferably 0.2 M.
  • the pretreatment is performed for 1 to 46 hours, preferably 5 to 22 hours, more preferably 8 to 10 hours; and the pretreatment temperature is 25 ⁇ 2 ° C;
  • the rotational speed of the treatment is: 110 ⁇ 20 rpm, preferably 110 ⁇ 10 rpm.
  • cryopreservation is cryopreservation.
  • the method further comprises: separating the pretreated cells; preferably, separating the pretreated cells by centrifugation; or
  • the steps include:
  • cell incubation incubation at 0 to 4 ° C, 110 ⁇ 20 rpm, preferably 110 ⁇ 10 rpm for 45 ⁇ 10min, preferably 45 ⁇ 5min;
  • the medium comprises: MS medium, 30 ⁇ 5 g/L sucrose, 1.0 ⁇ 0.2 mg/L 6-BA, 0.5 ⁇ 0.1 mg/L NAA+100 ⁇ 10 mg/L inositol.
  • the medium is a liquid medium.
  • a method of rapidly assessing cell viability of a plant cell comprising:
  • the plant cell is a mangosteen callus cell.
  • the higher the cell viability preservation rate the better the cell viability.
  • the cell viability preservation rate is higher than 30%, higher than 40%, higher than 50% or higher.
  • the detection result of 2,3,4-trichlorotetrazolium chloride is also considered in combination with the determination of the cell viability preservation rate as described above.
  • the present inventors used a Viable Cell Monitor as a tool for rapidly evaluating the effect of cryopreservation of cells, and evaluated the effect of cryoprotectants on cell preservation by changes in the capacitance of living cells before and after cell freezing and resuscitation.
  • the use of living cell electrodes can quickly and accurately characterize the concentration of living cells.
  • the effect of freezing methods on cell preservation can be quickly evaluated.
  • the inventors optimized the cryopreservation method of plant cells and found the optimum conditions and methods for cryopreservation.
  • the existing methods for evaluating cell viability after cryopreservation of cells are mainly methods such as restoration of growth and measurement of TTC value.
  • the recovery growth method restores the growth cell survival rate after resuscitating the cells, but the plant cells need to grow for a longer period of time, usually three weeks or more, and some cells even last up to 2 months, which makes the plant cells
  • the establishment of an optimized preservation method takes a long time.
  • the TTC method mainly uses the intracellular dehydrogenase to reduce the red form of the formamidine substance to make the cells develop color, which is used to detect the activity of mitochondria in the cells, but this method is difficult to distinguish the damage of the cell membrane during the cryopreservation process.
  • the dead cells cause a significant increase in the unreliability of the measured data.
  • the existing methods for evaluating cryopreservation of cells are too long and have relatively low sensitivity, which is not conducive to the establishment of cell preservation methods.
  • the invention utilizes a living cell sensor to detect the living cell capacitance value of the plant cells before and after cryopreservation, thereby obtaining the vigor preservation rate of the plant cells after cryopreservation.
  • the type of pretreatment agent, the pretreatment time and the type of cryoprotectant in the cell cryopreservation method can be rapidly screened, and the cell freezing method can be quickly evaluated to improve the survival rate of the plant cell after cryopreservation, and the operation is simple. At the same time, it shortens the time and improves efficiency.
  • the living cell sensor is, for example, Model 2801 or 220 of Aber Corporation.
  • the present inventors conducted various optimizations of the method of refrigerating plant cells by using the established living cell sensor to determine the activity of plant cells after cryopreservation, including cryoprotectants and freezing conditions. Operating procedures, etc.
  • Existing osmotic cryoprotectants including one type that can penetrate into cells, generally small molecules, mainly including glycerin, DMSO, ethylene glycol, propylene glycol, acetamide, methanol, etc.; and a type of non-permeate freezing Protecting agents, they can not penetrate into cells, usually macromolecular substances, mainly including polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, dextran, albumin and hydroxyethyl starch.
  • PVP polyvinylpyrrolidone
  • the present inventors adopt a living cell sensor to rapidly evaluate the cell viability in the process of rapid cryopreservation of Mangosteen callus cells, and the present invention utilizes a living cell sensor to cryopreservation of cells.
  • the viable cell capacitance values are detected before and after, so that the survival rate of the living cells after the cryopreservation method can be quickly obtained, and the effect of the specific preservation method can be evaluated in a short time, so that the cryopreservation method of the plant cells can be established quickly. Reduce time and increase efficiency.
  • a method for improving cell viability of cryopreserved cells comprising: (1) pretreating plant cells in a logarithmic growth phase with 0.15-0.3 M sucrose as a pretreatment agent (2) adding cells to a medium containing a cryoprotectant for cryopreservation; the cryoprotectant is DMSO and sucrose, and the final concentration in the medium is: DMSO: 8-12% (w /v); sucrose: 8-12% (w/v); more preferably, the final concentration in the medium is: DMSO: 9-11% (w/v); sucrose: 9-11% (w/ v).
  • the method further comprises: measuring and recording the capacitance value A 1 on the pretreated cells; and in the step (2), after the cryopreservation, further comprising: performing cell resuscitation on the plant cells, using the living cell sensing
  • the Viable Cell Monitor measures and records the capacitance value A 2 ; the cell's basic capacitance value is A 3 ; and the cell viability preservation rate is obtained by the formula (I):
  • the cell viability of the plant cells was evaluated based on the obtained cell viability preservation rate.
  • the plant cells of the invention may be a variety of plant cells, including their callus cells.
  • the plant cell is a Luo Han Guo cell; preferably a Mangosteen callus cell.
  • the inventors have optimized the method of pretreatment by measurement and analysis.
  • the sucrose is present in a liquid medium, and plant cells in a logarithmic growth phase are added to the liquid medium for pretreatment.
  • the concentration of the sucrose is 0.18 to 0.22 M; more preferably 0.2 M at the time of pretreatment.
  • the method further comprises: separating the pretreated cells; more preferably, separating the pretreated cells by centrifugation
  • the pretreatment time is preferably from 1 to 46 hours, preferably from 5 to 22 hours, more preferably from 8 to 10 hours.
  • the pretreatment temperature is 25 ⁇ 2 ° C; the pretreatment rotation speed is: 110 ⁇ 20 rpm, preferably 110 ⁇ 10 rpm.
  • the step of performing cryopreservation includes: (i) cell incubation: at 0 to 4 ° C, 110 ⁇ 20 rpm (preferably 110 ⁇ 10 rpm) Incubate for 45 ⁇ 10 min (preferably 45 ⁇ 5 min); (ii) store at 40 ⁇ 10 min (preferably 40 ⁇ 5 min) at -20 ⁇ 2 ° C, then at -80 ° C ⁇ 10 ° C ( Preferably stored at -80 ° C ⁇ 5 ° C).
  • Lo Han Guo Suspension Cells The embryos of Siraitia grosvenorii were selected to induce embryogenic loose calli. After being subcultured and screened, good and uniform callus was obtained, which was inoculated into liquid medium for suspension culture. , to obtain a state of the same suspension cell line.
  • Semi-solid medium MS medium + 30 g / L sucrose + 1.0 mg / L 6 - BA + 0.5 mg / L NAA + 100 mg / L inositol + 4.6 g / L agar.
  • Liquid medium MS medium + 30 g / L sucrose + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 100 mg / L inositol.
  • Pretreatment liquid liquid medium + sucrose (0.2 ⁇ 0.5M) / trehalose (0.2 ⁇ 0.5M).
  • Live cell sensor live cell concentration analyzer, obtained from Aber: 2801-00
  • -80 ° C ultra-low temperature refrigerator rotary shaker.
  • DMSO dimethyl sulfoxide
  • sucrose sucrose
  • sorbitol sucrose
  • trehalose Shanghai Aladdin Biotechnology Co., Ltd.
  • Centrifuge or sediment a certain amount of cells in the logarithmic growth phase, remove the supernatant, and add an equal amount of pretreatment solution. Pretreatment at 25 ° C, 110 rpm for 18 h.
  • the pretreated cells were centrifuged, the supernatant was removed, and a cryoprotectant was added thereto, and the cells were incubated at 0 to 4 ° C and 110 rpm for 45 minutes.
  • the incubated cells were firstly cooled at -20 ° C for 40 min, and then placed in a -80 ° C ultra-low temperature refrigerator to cool down.
  • the frozen cells were thawed under a water bath condition of 39 ° C, usually 2-3 min. After the ice in the centrifuge tube is completely dissolved, the cryoprotectant having a certain toxicity is quickly removed after centrifugation, and washed three times with the pretreatment liquid.
  • the viable cell capacitance was measured by a living cell sensor.
  • the viable cell capacitance value of the pretreated cells was recorded as A 1
  • the viable cell capacitance value after resuscitation was recorded as A 2
  • the cell's basic capacitance value was recorded as A 3 (capacitance value of complete cell death).
  • Cell viability preservation rate (A 2 - A 1 ) / (A 3 - A 1 ).
  • the basic capacitance value of the cells the cell suspension was boiled in boiling water for 1 h, and then ultrasonically disrupted for 40 min, and the measured capacitance value was the basic capacitance value.
  • the cells in the logarithmic growth phase were used as the initial cells, and the cells were diluted 2, 4, 6, and 8 times, respectively, and the capacitance value was measured by a living cell sensor.
  • the living cell capacitance value has a good linear relationship with the cell concentration.
  • the cell capacitance value in the logarithmic growth phase is about 100pF/cm, and only one base capacitance value is about 7pF/cm in the solution of the medium. After the cells are completely lethal, the capacitance value is not much different from the basic capacitance value of the medium. This was also demonstrated in subsequent experiments.
  • Glycerin, DMSO, sucrose, sorbitol and the like which have been used as a cryoprotectant.
  • glycerin and DMSO are osmotic cryoprotectants
  • sucrose and sorbitol are non-permeable cryoprotectants.
  • the cell activity of the cryoprotectant is much higher than that of the cells without the cryoprotectant, both in terms of the viable cell capacitance value and the cell mitochondrial activity.
  • the cell viability preservation rate was greatly improved compared with the control group.
  • the living cell capacitance value there is a good correspondence between the living cell capacitance value and the TTC value. Therefore, the cell viability preservation rate can reflect the viability of the cells after cryopreservation.
  • the viable cell capacitance and TTC values at various levels of DMSO, sucrose, and sorbitol were statistically analyzed, and the TTC values and viable cell capacitance values at various levels were comprehensively scored.
  • the results of the effects of each level of cryoprotectant on the activity of cells after cryopreservation are shown in Table 2.
  • TTC value score TTC value / maximum TTC value ⁇ 10;
  • Vitality preservation rate score vitality preservation rate / maximum vitality preservation rate ⁇ 10;
  • 10% DMSO was better than 5% DMSO after cryopreservation.
  • 10% content is the best for cell cryopreservation.
  • cryoprotectant that are well preserved for cryopreservation of cells are: cell growth medium (liquid medium) + 10% DMSO + 10% sucrose, compared with cells pretreated without cryoprotectant. Under these conditions, the viability preservation rate and mitochondrial activity (TTC value) of the cells increased by 224.43% and 372.16%, respectively.
  • Example 3 the effect of different types of pretreatment agents on cell cryopreservation
  • the type of pretreatment agent and the concentration of the pretreatment agent have different effects on the cryopreservation of the cells.
  • the pretreatment agent concentration is too high, although the dehydration effect is better, the high concentration of the pretreatment agent damages the cells.
  • sucrose 0.2M, 0.35M, 0.5M, and trehalose 0.2M, 0.35M, and 0.5M were selected for investigation.
  • the control was directly subjected to cryopreservation without pretreatment.
  • the freezing time was: -20 ° C for 40 min, then stored at -80 ° C for 24 h; frozen medium: cell growth medium (liquid medium) + 10% DMSO + 10% sucrose; Determination.
  • TTC value score TTC value / maximum TTC value *
  • vitality preservation rate score vitality preservation rate / maximum vitality preservation rate *
  • comprehensive score TTC value score + vitality preservation rate score.
  • the experimental results showed that the cell growth medium (liquid medium) + 10% DMSO + 10% sucrose as a cryoprotectant, the cells were pretreated with sucrose 0.2M pretreatment agent for ultra-low temperature preservation, compared with the control, cell viability
  • the preservation rate and mitochondrial activity (TTC value) were greatly improved, and the viability preservation rate and cell activity were increased by 29.18.% and 36.72%, respectively.
  • the effect of pretreatment time on the activity of the cells after cryopreservation is similar to the concentration of the pretreatment agent. If the cells are treated for a long time in an environment with a certain osmotic pressure, the cells may be excessively dehydrated, thus causing the cells to The activity in the pretreatment stage is greatly reduced, thereby affecting the activity of the cells after cryopreservation.
  • the pretreatment time was 0h, 1h, 2h, 5h, 9h, 22h and 46h with sucrose 0.2M as pretreatment agent.
  • the storage conditions were: -20 ° C for 40 min, then stored at -80 ° C for 24 h; frozen medium: liquid medium + 10% DMSO + 10% sucrose, then into Line recovery assay.
  • the experimental results show that the cell growth medium (liquid medium) + 10% DMSO + 10% sucrose as a cryoprotectant, the cells are pretreated with sucrose 0.2M pretreatment agent for 9-22h hours, and the ultra-low temperature preservation and preservation effect is relatively ideal. Among them, the ultra-low temperature preservation and cell preservation effect were best after 9h, and the viability preservation rate and mitochondrial activity (TTC value) of the cells were greatly improved compared with the control (pretreatment 0h).

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Abstract

本发明涉及活细胞传感仪在植物细胞低温保藏中的应用。首次将活细胞传感仪作为一种快速评价细胞冻存效果的方法，通过细胞冷冻和复苏前后活细胞电容值的变化来评价冷冻保护剂对细胞保存的效果。采用活细胞电极则可以快速、准确地表征活细胞的浓度。将活细胞电容值与2,3,4-氯化三苯基四氮唑(TTC)的检测结果结合，可达到快速定量评价冷冻方法对细胞保存的效果，克服了以往植物细胞保藏方法的低效性和无定量性。本发明的方法与传统方法相比，细胞低温保藏综合得分可以提高5倍或以上。

Description

活细胞传感仪在植物细胞低温保藏中的应用 技术领域
本发明属于细胞检测领域，更具体地，本发明涉及活细胞传感仪在植物细胞低温保藏中的应用。
背景技术
现有的植物细胞系的保种主要通过以下两种方式。第一种是在生长培养基中进行继代保存，另一种则是对细胞进行低温冷冻保藏。通过长期继代而保存细胞存在费时费力、成本昂贵，并且会出现植物细胞特性消失等缺点。而细胞在超低温的条件下时，会停止内部的一系列代谢活动，并能够保持细胞自身的特性，大大降低细胞特性消失的风险。而植物细胞超低温保藏中，冷冻保藏的方法则对于细胞能够保存成功起到决定性作用。由于植物细胞中含有大量的水分，给冷冻保存带来困难。因此需要选择适合的冷冻保护剂，以及优化适合的冷冻保存方法，以最有效地控制细胞内含水以及冷冻过程中对细胞的损伤，以期可以在细胞冷冻保存后获得更高的细胞活性。
植物细胞在冷冻保藏过程中会由于内部或者外部水结成冰晶而造成细胞破裂，以往，均是采用细胞线粒体活性测定的方法来间接反映细胞在冷冻保藏后的活性情况。但是，这种测定操作烦琐，处理条件复杂，耗时长，在实际操作存在困难。
活细胞传感仪是利用电容法测量细胞溶液中的活细胞浓度。细胞溶液中存在的交变电场会使其中的离子发生迁移，而活细胞具有完整的绝缘性细胞膜，其中的离子受到束缚导致细胞膜极化，这使得细胞成为一个个微小的电容器。测量得到的电容值与溶液中的细胞量存在良好的线性关系。目前活细胞传感仪常被应用于发酵过程中测定发酵液中细胞量，尚未被应用于细胞冷冻保存中。
发明内容
本发明的目的在于提供活细胞传感仪在植物细胞低温保藏中的应用。
在本发明的第一方面，提供一种提高冷冻保存细胞的细胞活力的方法，所述方法包括：
(1)用0.15～0.3M蔗糖作为预处理剂对处于对数生长期的植物细胞进行预处理；
(2)将细胞加入到含有冷冻保护剂的培养基中，进行低温保存；所述的冷冻保护剂为DMSO和蔗糖，在培养基中的终浓度为：
DMSO：8～12％(w/v)；更佳地为9～11％(w/v)；
蔗糖：8～12％(w/v)；更佳地为9～11％(w/v)。
在一个优选例中，所述方法的步骤(1)中，还包括：对预处理后的细胞测定并记录电容值A1；
步骤(2)中，冷冻保存后，还包括：对植物细胞进行细胞复苏，利用活细胞传感仪测定并记录电容值A2；
以细胞的基础电容值为A3；以式(I)获得细胞活力保存率：
细胞活力保存率＝(A2-A1)/(A3-A1)         (I)；
根据获得的细胞活力保存率，评价植物细胞的细胞活力。
在另一优选例中，所述的植物细胞是罗汉果植物细胞；较佳地是罗汉果愈伤组织细胞。
在另一优选例中，所述方法的步骤(1)中，所述的蔗糖存在于液体培养基中，将处于对数生长期的植物细胞加入到液体培养基中，进行预处理。
在另一优选例中，所述的方法的步骤(1)中，以蔗糖作为预处理剂时，所述的蔗糖的浓度为0.18～0.22M；更佳地为0.2M。
在另一优选例中，所述方法中，进行预处理的时间为1～46小时，较佳地5～22小时，更佳地8-10小时；预处理的温度为25±2℃；预处理的转速为：110±20rpm，较佳地110±10rpm。
在另一优选例中，所述的低温保存是冻存。
在另一优选例中，所述方法的步骤(1)后，还包括：分离经过预处理的细胞；较佳地，通过离心分离预处理的细胞；或
步骤(2)中，将细胞加入到含有冷冻保护剂的培养基中后，进行低温保存的 步骤包括：
(i)细胞孵育：在0～4℃，110±20rpm，较佳地110±10rpm条件下孵育45±10min，较佳地45±5min；
(ii)先在-20±2℃下保存40±10min，较佳地40±5min，再放到-80℃±10℃，较佳地-80℃±5℃保存。
在另一优选例中，所述方法中，所述的培养基含有：MS培养基，30±5g/L蔗糖，1.0±0.2mg/L 6-BA，0.5±0.1mg/L NAA+100±10mg/L肌醇。较佳地，所述的培养基为液体培养基。
在本发明的另一方面，提供一种快速评价植物细胞的细胞活力的方法，所述方法包括：
(a)将植物细胞进行冷冻保存；冷冻保存前测定并记录电容值A1；
(b)冷冻保存后，对植物细胞进行细胞复苏，利用活细胞传感仪测定并记录电容值A2；
(c)以细胞的基础电容值为A3；以式(I)获得细胞活力保存率：
细胞活力保存率＝(A2-A1)/(A3-A1)          (I)；
(d)根据(c)获得的细胞活力保存率，评价植物细胞的细胞活力。
在一个优选例中，所述的植物细胞是罗汉果愈伤组织细胞。
在另一优选例中，细胞活力保存率越高，则细胞活力越好。例如，所述的细胞活力保存率高于30％，高于40％，高于50％或更高。
在另一优选例中，在如上确定细胞活力保存率的同时，还结合考虑2,3,4-氯化三苯基四氮唑(TTC)的检测结果。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、活细胞电容值与细胞浓度的线性关系。
具体实施方式
本发明人首次将活细胞传感仪(Viable Cell Monitor)作为一种快速评价细胞冻存效果的工具，通过细胞冷冻和复苏前后活细胞电容值的变化来评价冷冻保护剂对细胞保存的效果。采用活细胞电极则可以快速、准确地表征活细胞的浓度，将活细胞电容值与TTC的检测结果结合起来，就可以快速评价冷冻方法对细胞保存的效果。
并且，根据应用活细胞传感仪的测定及分析，本发明人优化了植物细胞的冷冻保存方法，找到了最佳的进行冷冻保存的条件和方法。
活细胞传感仪应用于低温冷藏细胞评估
现有的评价细胞低温保藏后细胞活率的方法主要是通过恢复生长、TTC值测定等方法。恢复生长法是通过将细胞复苏后，使其恢复生长统计细胞活率，但是植物细胞恢复生长需要一个较长的周期，一般三周以上，某些细胞甚至长达2个月，这使得植物细胞最优化保藏方法的建立需要耗费很长的时间。而TTC方法则主要是利用细胞内脱氢酶还原成红色的甲臜类物质使细胞显色，用于检测细胞内线粒体的活性，但是这种方法难于区分在冷冻保藏过程中由于细胞膜的破损等而死亡的细胞，造成测定数据的不可靠性大幅度增加。
现有的评价细胞低温保藏的方法耗时太长，敏感性比较低，不利于细胞保存方法的建立。本发明利用活细胞传感仪，能够对植物细胞低温保藏前后的活细胞电容值进行检测，从而获得的低温保藏方法对植物细胞低温保藏后的的活力保存率。可以将细胞冻存方法中的预处理剂种类、预处理时间和冷冻保护剂的种类进行快速的筛选，通过快速地对细胞冷冻方法进行评价，提高植物细胞低温保藏后的存活率，操作简单，同时缩短了时间，提高效率。所述的活细胞传感仪例如Aber公司的2801-00或220型。
植物细胞低温冷藏方法的优化
利用前述建立的活细胞传感仪测定植物细胞低温冷藏后活性的方法，本发明人进行了植物细胞冷藏方法的多方面优化，包括冷冻保护剂、冷冻条件。操作程序等。
现有的渗透性冷冻保护剂，包括一类可以渗透到细胞内，一般是一些小分子物质，主要包括甘油、DMSO、乙二醇、丙二醇、乙酰胺、甲醇等；以及一类非渗透性冷冻保护剂，它们不能渗透到细胞内，一般是些大分子物质，主要包括聚乙烯吡咯烷酮(PVP)、蔗糖、聚乙二醇、葡聚糖、白蛋白以及羟乙基淀粉等。
将渗透和非渗透性冷冻保护剂混合使用，除了可以对细胞起到脱水的作用，可以较好的控制细胞内外冰晶的形成以及冰晶的形状，防止有尖的冰晶生成并刺伤细胞，从而对细胞起到保护的作用。因此，本发明人经过反复研究，选择液体培养基+DMSO+蔗糖、同时含有渗透和非渗透性冷冻保护剂的制剂，应用于进行植物细胞的低温冷藏保护。
在本发明的实施例中，本发明人采用活细胞传感仪进行罗汉果愈伤组织细胞快速低温冷冻保藏过程中细胞存活率的快速评价体系，本发明利用活细胞传感仪，对细胞低温保藏前后的活细胞电容值进行检测，从而快速得到低温保藏方法后活细胞存活率，实现在较短的时间内对特定保藏方法的效果进行评价，从而能够较快的建立植物细胞的低温保藏方法，缩短时间，提高效率。
基于本发明人的新发现，提供一种提高冷冻保存细胞的细胞活力的方法，所述方法包括：(1)用0.15～0.3M蔗糖作为预处理剂对处于对数生长期的植物细胞进行预处理；(2)将细胞加入到含有冷冻保护剂的培养基中，进行低温保存；所述的冷冻保护剂为DMSO和蔗糖，在培养基中的终浓度为：DMSO：8～12％(w/v)；蔗糖：8～12％(w/v)；更佳地，在培养基中的终浓度为：DMSO：9～11％(w/v)；蔗糖：9～11％(w/v)。
在冷冻处理过程，可以进行细胞活力的测定，以随时了解到细胞活性的变化。因此，步骤(1)中，还包括：对预处理后的细胞测定并记录电容值A1；步骤(2)中，冷冻保存后，还包括：对植物细胞进行细胞复苏，利用活细胞传感仪(Viable Cell Monitor)测定并记录电容值A2；以细胞的基础电容值为A3；以式(I)获得细胞活力保存率：
细胞活力保存率＝(A2-A1)/(A3-A1)           (I)；
根据获得的细胞活力保存率，评价植物细胞的细胞活力。
本发明的植物细胞可以是多种植物细胞，包括它们的愈伤组织细胞。作为 本发明的优选方式，所述的植物细胞是罗汉果细胞；较佳地是罗汉果愈伤组织细胞。
本发明人经过测定分析，优化了预处理的方法。将所述的蔗糖存在于液体培养基中，将处于对数生长期的植物细胞加入到液体培养基中，进行预处理。较佳地，预处理时，所述的蔗糖的浓度为0.18～0.22M；更佳地为0.2M。较佳地，在预处理后，还包括：分离经过预处理的细胞；更佳地，通过离心分离预处理的细胞
本发明人还分析了预处理的时间，结果发现，预处理的时间为1～46小时，较佳地5～22小时，更佳地8-10小时是较为理想的。
作为本发明的优选方式，预处理的温度为25±2℃；预处理的转速为：110±20rpm，较佳地110±10rpm。
作为本发明的优选方式，将细胞加入到含有冷冻保护剂的培养基中后，进行低温保存的步骤包括：(i)细胞孵育：在0～4℃，110±20rpm(较佳地110±10rpm)条件下孵育45±10min(较佳地45±5min)；(ii)先在-20±2℃下保存40±10min(较佳地40±5min)，再放到-80℃±10℃(较佳地-80℃±5℃)保存。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
1.1菌种和培养基
菌种：罗汉果悬浮细胞：选取罗汉果的种胚，诱导出胚性松散的愈伤组织，经过继代和筛选获得生长状态良好、均匀的愈伤组织，将其接种到液体培养基中进行悬浮培养，获得状态均一的悬浮细胞系。
半固体培养基：MS培养基+30g/L蔗糖+1.0mg/L 6-BA+0.5mg/LNAA+100mg/L肌醇+4.6g/L琼脂。
液体培养基：MS培养基+30g/L蔗糖+1.0mg/L 6-BA+0.5mg/LNAA+100mg/L肌醇。
预处理液：液体培养基+蔗糖(0.2～0.5M)/海藻糖(0.2～0.5M)。
含有冷冻保护剂的培养基：液体培养基+DMSO(5％～10％)+蔗糖(5％～10％)/山梨醇(5％～10％)。
1.2仪器和试剂
仪器：活细胞传感仪(活细胞浓度分析仪，获自Aber公司：2801-00)、-80℃超低温冰箱、旋转式摇床。
试剂：DMSO(二甲基亚砜)、蔗糖、山梨醇、海藻糖：上海阿拉丁生化科技股份有限公司。
1.3细胞的超低温保藏方法
1.3.1预处理
取一定量处于对数生长期的细胞进行离心(或沉降)，去掉上清后，加入等量的预处理液。在25℃，110rpm的条件下预处理18h。
1.3.2孵育
将预处理过的细胞进行离心，去掉上清后，加入冷冻保护剂，在0～4℃，110rpm条件下孵育45min。
1.3.3冷冻
将孵育后的细胞先在-20℃降温40min，再放到-80℃超低温冰箱降温。
1.3.4复苏
将冷冻过的细胞在39℃的水浴条件下进行化冻，一般2-3min为宜。至离心管中的冰完全溶解后，离心后迅速将具有一定毒性的冷冻保护剂去掉，用预处理液清洗三次。
1.4测定方法
(1)细胞活力评价方法
悬浮细胞混匀后用活细胞传感仪测定活细胞电容值。预处理后的细胞的活细胞电容值记为A1，复苏后的活细胞电容值记为A2，细胞的基础电容值记为A3(细胞完全死亡的电容值)。细胞活力保存率＝(A2-A1)/(A3-A1)。
细胞的基础电容值：将细胞悬液放在沸水中煮沸1h后，再超声破碎40min，测的电容值为基础电容值。
(2)细胞线粒体活性测定
悬浮细胞经8μm滤膜抽滤后，取100mg鲜重细胞，分别加入2.5mL 0.4％的2,3,4-氯化三苯基四氮唑(TTC)溶液和2.5mL 0.05mol/L(pH 7.5)的Na2HPO4-NaH2PO4的磷酸缓冲液，混匀后25℃暗处理16h。暗培养后离心去上清液，蒸馏水洗涤3次，加入4mL甲醇，置于60℃水浴50min，期间用枪头轻轻吹打两次，至细胞脱至无色。室温静置后，4000rpm离心5min后取上清液，在485nm下测定吸光度，间接反映细胞活力。
实施例1、活细胞电容值与细胞浓度的线性关系
以对数生长期的细胞作为初始细胞，分别将细胞稀释2、4、6、8倍后，利用活细胞传感仪测定电容值。
结果如图1，从图1可以看出，活细胞电容值与细胞浓度具有很好的线性关系。对数生长期的细胞电容值在100pF/cm左右，而只有培养基的溶液中存在一个基础电容值在7pF/cm左右，将细胞完全致死后，电容值与培养基基础电容值相差不大，这个在后续的实验中也得到了证明。
从图1的结果可以得出结论，活细胞电容值能够很好地表征活细胞浓度，这也为后续的实验提供了基础。
实施例2、不同类型的冷冻保护剂对细胞低温保藏的影响
已经被用作冷冻保护剂的有甘油、DMSO、蔗糖、山梨醇等。其中，甘油和DMSO为渗透性冷冻保护剂，蔗糖和山梨醇是非渗透性冷冻保护剂。本实施例中探讨如何选择冷冻保护剂，选择怎样的浓度可以获得最为优异的保护效果。
采用不同浓度的DMSO(5％、10％)分别和蔗糖(5％、10％)、山梨醇(5％、10％) 进行混合作为冷冻保护剂进行研究，将细胞用含有0.35M蔗糖的液体培养基进行预处理18h后，将预处理过的细胞进行离心，去掉上清后，加入冷冻保护剂，在0～4℃，110rpm条件下孵育45min。将孵育后的细胞先在-20℃降温40min，再放到-80℃超低温冰箱降温24h后，测定细胞经过低温保藏后的活力。分组情况如表1，其中9组是经过预处理但是不加冷冻保护剂的细胞。
冷冻保护剂对细胞低温保藏活力的影响结果如表1。
表1、冷冻保护剂对细胞低温保藏活力的影响
Figure PCTCN2017112299-appb-000001
从实验结果可以看出，不管是在活细胞电容值还是细胞线粒体活性上来看，加入冷冻保护剂的细胞活性都远远高于不添加冷冻保护剂的细胞。从活细胞电容值水平上，细胞活力保存率较对照组都有很大的提高。而且活细胞电容值和TTC值也有一个较好的对应关系。因此，细胞活力保存率可以反应细胞在冻存后的活力情况。
将DMSO、蔗糖、山梨醇各个水平下活细胞电容值和TTC值进行统计分析，并将各个水平下的TTC值和活细胞电容值进行综合评分。各水平冷冻保护剂对细胞低温保藏后活性的影响结果见表2。
表2、各水平冷冻保护剂对细胞低温保藏后活性的影响
冷冻保护剂 活力保存率得分 TTC值得分 综合得分
5％蔗糖 31.15％ 0.506 13.215
10％蔗糖 42.98％ 0.847 20.000
5％山梨醇 31.20％ 0.652 14.951
10％山梨醇 40.65％ 0.846 19.441
5％DMSO 35.90％ 0.633 15.825
10％DMSO 37.09％ 0.792 17.978
注：TTC值得分＝TTC值/最大TTC值×10；
活力保存率得分＝活力保存率/最大活力保存率×10；
综合得分＝TTC值得分+活力保存率得分。
从表2的结果可以看出，10％含量的DMSO对细胞低温保藏后的效果要优于5％DMSO。而对于山梨醇和蔗糖，不管是活细胞电容值还是TTC值，10％的含量对于细胞低温保藏的效果都是最好的。
经过综合评分，确定对细胞低温保藏较好的冷冻保护剂成分为：细胞生长培养基(液体培养基)+10％DMSO+10％蔗糖，与经过预处理但是不加冷冻保护剂的细胞相比，此条件下细胞的活力保存率和线粒体活性(TTC值)分别提高了224.43％、372.16％。
实施例3、不同类型的预处理剂对细胞低温保藏的影响
预处理剂的种类以及预处理剂的浓度不同，对于细胞低温保藏也有影响，预处理剂浓度过高时，虽然脱水效果比较好，但是高浓度的预处理剂对细胞的损伤也比较大。在前面实验的基础上，选择蔗糖0.2M、0.35M、0.5M以及海藻糖0.2M、0.35M、0.5M进行考察，对照是不进行预处理，直接进行冻存的细胞。冻存时间均为：-20℃放置40min后，放入-80℃进行保存24h；冻存培养基均为：细胞生长培养基(液体培养基)+10％DMSO+10％蔗糖；之后进行复苏测定。
实验结果见表3。
表3、预处理剂种类及浓度对细胞低温保藏后活性的影响
Figure PCTCN2017112299-appb-000002
Figure PCTCN2017112299-appb-000003
注：TTC值得分＝TTC值/最大TTC值*10，活力保存率得分＝活力保存率/最大活力保存率*10，综合得分＝TTC值得分+活力保存率得分。
实验结果表明，以细胞生长培养基(液体培养基)+10％DMSO+10％蔗糖作为冷冻保护剂，细胞经过蔗糖0.2M预处理剂预处理后进行超低温保藏，与对照相比，细胞的活力保存率和线粒体活性(TTC值)均有大幅度的提高，活力保存率和细胞活性分别提高了29.18.％、36.72％。
实施例4、预处理时间对细胞低温保藏的影响
预处理时间对细胞冷冻保藏后活性的影响与预处理剂的浓度原理相似，若细胞在具有一定渗透压的环境下处理时间过长时，有可能造成细胞的过度失水，这样就造成细胞在预处理阶段的活性大大降低，从而影响细胞冻存复苏后的活性。在前面实验的基础上，以蔗糖0.2M为预处理剂，对预处理时间0h、1h、2h、5h、9h、22h、46h进行考察。冻存条件为：-20℃放置40min后，放入-80℃进行保存24h；冻存培养基均为：液体培养基+10％DMSO+10％蔗糖，之后进 行复苏测定。
实验结果见4。
表4、预处理时间对细胞低温保藏后活性的影响
Figure PCTCN2017112299-appb-000004
实验结果表明，以细胞生长培养基(液体培养基)+10％DMSO+10％蔗糖作为冷冻保护剂，细胞经过蔗糖0.2M预处理剂预处理9-22h小时后进行超低温保藏、保存效果相对理想，其中以9h后进行超低温保藏、细胞保存效果最好，与对照(预处理0h)相比，细胞的活力保存率和线粒体活性(TTC值)均有大幅度的提高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种提高冷冻保存细胞的细胞活力的方法，其特征在于，所述方法包括：
    (1)用0.15～0.3M蔗糖作为预处理剂对处于对数生长期的植物细胞进行预处理；
    (2)将细胞加入到含有冷冻保护剂的培养基中，进行低温保存；所述的冷冻保护剂为DMSO和蔗糖，在培养基中的终浓度为：
    DMSO：8～12％(w/v)；
    蔗糖：8～12％(w/v)。
  2. 如权利要求1所述的方法，其特征在于，步骤(1)中，还包括：对预处理后的细胞测定并记录电容值A1；
    步骤(2)中，冷冻保存后，还包括：对植物细胞进行细胞复苏，利用活细胞传感仪测定并记录电容值A2；
    以细胞的基础电容值为A3；以式(I)获得细胞活力保存率：
    细胞活力保存率＝(A2-A1)/(A3-A1)      (I)；
    根据获得的细胞活力保存率，评价植物细胞的细胞活力。
  3. 如权利要求1所述的方法，其特征在于，所述的植物细胞是罗汉果植物细胞；较佳地是罗汉果愈伤组织细胞。
  4. 如权利要求1所述的方法，其特征在于，步骤(1)中，所述的蔗糖存在于液体培养基中，将处于对数生长期的植物细胞加入到液体培养基中，进行预处理。
  5. 如权利要求1所述的方法，其特征在于，步骤(1)中，以蔗糖作为预处理剂时，所述的蔗糖的浓度为0.18～0.22M；更佳地为0.2M。
  6. 如权利要求1所述的方法，其特征在于，进行预处理的时间为1～46小时，较佳地5～22小时，更佳地8-10小时；预处理的温度为25±2℃；预处 理的转速为：110±20rpm。
  7. 如权利要求1所述的方法，其特征在于，步骤(1)后，还包括：分离经过预处理的细胞；较佳地，通过离心分离预处理的细胞；或
    步骤(2)中，将细胞加入到含有冷冻保护剂的培养基中后，进行低温保存的步骤包括：
    (i)细胞孵育：在0～4℃，110±20rpm条件下孵育45±10min；
    (ii)先在-20±2℃下保存40±10min，再放到-80℃±10℃保存。
  8. 如权利要求1所述的方法，其特征在于，所述的培养基含有：MS培养基，30±5g/L蔗糖，1.0±0.2mg/L 6-BA，0.5±0.1mg/L NAA+100±10mg/L肌醇。
  9. 一种快速评价植物细胞的细胞活力的方法，其特征在于，所述方法包括：
    (a)将植物细胞进行冷冻保存；冷冻保存前测定并记录电容值A1；
    (b)冷冻保存后，对植物细胞进行细胞复苏，利用活细胞传感仪测定并记录电容值A2；
    (c)以细胞的基础电容值为A3；以式(I)获得细胞活力保存率：
    细胞活力保存率＝(A2-A1)/(A3-A1)        (I)；
    (d)根据(c)获得的细胞活力保存率，评价植物细胞的细胞活力。
  10. 如权利要求9所述的方法，其特征在于，细胞活力保存率越高，则细胞活力越好。
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