CN105316277A - Three-dimensional culture method of adherent cells - Google Patents

Three-dimensional culture method of adherent cells Download PDF

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CN105316277A
CN105316277A CN201510690303.5A CN201510690303A CN105316277A CN 105316277 A CN105316277 A CN 105316277A CN 201510690303 A CN201510690303 A CN 201510690303A CN 105316277 A CN105316277 A CN 105316277A
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culture
cell
cells
magnetic field
beads
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CN201510690303.5A
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曾宪卓
曾明哲
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深圳华毓造血干细胞研究有限公司
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Abstract

The invention provides a three-dimensional culture method of adherent cells. The three-dimensional culture method includes: acquiring magnetic beads with cell adherence; placing the magnetic beads as a co-culture medium and to-be-cultured cells in a magnetic field for culture. According to the method, the magnetic beads are used as the cell co-culture medium; compared with glucose or micro-bead culture media of synthetic polymer, in the process of cell culture, distribution of the magnetic beads can be changed by controlling and changing intensity of a magnetic field according to conditions such as space and density required at each culture period, and culture environment can be changed according to requirements of cell growth stage. The magnetic field is externally added to enable the magnetic beads to move, so that a mechanical stirring rod and an airlift stirring device can be replaced to enable a culture solution and cells to flow, so that damage to the cells caused by mechanical impact and shearing stress is reduced.

Description

贴壁性细胞的三维培养方法 Three-dimensional culture of adherent cells

技术领域 FIELD

[0001] 本发明属于细胞三维培养技术领域,具体涉及一种贴壁性细胞的三维培养方法。 [0001] The present invention belongs to the three-dimensional cell culture technology, particularly relates to a method for three-dimensional culture of cells attached to the wall.

背景技术 Background technique

[0002] 针对干细胞或者肿瘤细胞等贴壁性细胞采用二维培养方法,培养出来的细胞都是粘附在容器壁上的二维形态细胞,不利于促分化、细胞微环境控制、以及应用等方面的不足;因此多采用不同材料的三维结构载体与细胞形成细胞-三维载体复合的形态,在体外共同培养,使细胞能够在载体的三维立体空间结构中迀移、生长。 [0002] The two-dimensional culture method for tumor cells, or stem cells adherent cells, the cells are cultivated in two-dimensional morphology of the cells adhered to the container wall, it is not conducive to promoting differentiation, cell microenvironment control, and other applications deficiencies; therefore use more three-dimensional structure of the carrier and the cells of different materials forming cells - dimensional complex vector form, co-cultured in vitro, the cells can be shifted in Gan three-dimensional spatial structure of the carrier, the growth.

[0003] 而构建三维细胞培养的三维结构载体现有通常是两种,一种是三维细胞支架、另一种是微球载体。 [0003] constructed in a three-dimensional cell culture carrier prior three-dimensional structures are generally two types of three-dimensional cell scaffold, the other carrier is a microsphere. 其中,三维细胞支架的三维细胞培养方式是在培养容器内固设胶原、蚕丝蛋白、纤维蛋白、海藻酸盐、透明质酸、壳聚糖等材质制备的利于细胞亲和的支架体;微球载体是能适用于贴壁细胞生长的微珠,一般是由天然葡聚糖或者各种合成的聚合物组成。 Wherein the three-dimensional cell culture is a three-dimensional cell culture scaffold collagen fixed within the container, is conducive to cell affinity stent body prepared fibroin, fibrin, alginate, hyaluronic acid, chitosan and other materials; microspheres carrier is applicable to the growth of adherent cells attached to the beads, typically by a variety of natural or synthetic dextran polymer. 上述三维培养方式在实施的过程中,三维细胞支架一旦设定之后,支架本身不会再移动,培养相对比较静态;所以需要进行细胞动态环境培养时,多采用的是微球载体进行。 The three-dimensional culturing mode in the course of implementation, after the three-dimensional cell scaffold once set, will not move the stent itself, culture relatively static; when it is necessary to perform dynamic environment cultured cells, the use of the carrier is a microsphere.

[0004] 但采用微球载体进行三维培养时,微球载体预先根据细胞培养的密度和数量要求计算后添加至培养容器中,并用搅拌等方式保持其悬浮在培养液中;但是培养过程中除了细胞数量和密度增加之外,微球自身的构建的环境(微球自身密度等)也是不变的;而对于细胞培养来说,细胞在不同生长阶段(前期接种的细胞数量不多、需要微球相对密度大促进贴壁,中后期贴壁细胞数量增加后、相应降低微球密度促进其生长增殖)上需要不同的环境,微球载体也无法较好地满足动态环境的要求;并且,微球载体搅拌方式保持悬浮本身也增加了细胞培养的剪切应力,容易对细胞造成大的机械损伤,因此也无法达到实现良好的细胞三维动态培养环境,降低了细胞培养的品质。 [0004] When employing the microsphere carrier three-dimensional culture, microspheres were previously added according to the cell culture density and the number of requests calculated to the culture vessel, and with stirring, etc. held suspended in the culture medium; however culture process except outside the cell number and density increase, the microspheres constructed environment itself (self-density microspheres and the like) are also the same; and for cell culture, the cells were small number of cells at different growth stages (pre-inoculation, the micro required ball relative density promoting adherence, after increasing the number of late parietal cell paste, a corresponding decrease in the density microspheres promote proliferation) requires a different environment, the microsphere carriers can not satisfy the requirements of a dynamic environment; and micro ball carrier itself remain suspended stirred embodiment increases the shear stress of the cell culture, likely to cause mechanical damage to the large cells, and therefore can not achieve good dynamic three-dimensional cell culture environment, reducing the quality of the cell culture.

发明内容 SUMMARY

[0005] 本发明实施的目的在于克服现有微珠载体继续拧细胞三维培养的缺陷,提供一种适于细胞培养环境过度的贴壁性细胞的三维培养方法,及实现该方法的装置。 [0005] The object of the embodiment of the present invention is to overcome the prior carrier beads three-dimensional cell culture to continue screwing the defect, there is provided a method of three-dimensional culture adapted cell culture environment excessive adherent cells, and an apparatus implementing the method.

[0006] 为了实现上述发明目的,本发明实施例的技术方案如下: [0006] In order to achieve the above object, the technical solution of the embodiments of the present invention are as follows:

[0007] —种贴壁性细胞的三维培养方法,包括: [0007] - a three-dimensional culture of adherent cell types, comprising:

[0008] 获取细胞贴壁性的磁珠; [0008] Gets beads of adherent cells;

[0009] 以所述磁珠作为共培养介质与待培养细胞于磁场中进行培养。 [0009] In the co-culture medium as a magnetic bead to be cultured cells cultured in a magnetic field.

[0010] 采用本发明的上述方法,以磁珠作为细胞共培养介质,相比葡聚糖或合成聚合物的微珠培养介质,在细胞培养的过程中可以根据各培养时期所要求的空间、密度等条件,通过控制改变磁场的场强即可使磁珠的分布产生变化,从而可以根据细胞生长的阶段要求改变培养环境。 [0010] The above-described method of the present invention, magnetic beads as cell co-culture medium, the culture medium compared with dextran microbeads or synthetic polymers, in the course of the cell culture space may be required according to each culture period, density and other conditions, by controlling the magnetic field intensity to change the distribution of the magnetic beads so that a difference, thereby changing the culture environment may be required depending on the stage of cell growth. 而且,通过外加磁场来使磁珠进行运动,可以替代机械搅拌棒、气升式等搅拌使培养液和细胞流动,降低了机械冲击和剪切应力对细胞的伤害。 Further, by applying a magnetic field to make the movement of magnetic beads, can replace mechanical stirrer, airlift culture solution and the like is stirred cell flow, reduces the mechanical impact and shear stress damage to the cells.

附图说明 BRIEF DESCRIPTION

[0011] 下面将结合附图及实施例对本发明作进一步说明,附图中: [0011] The accompanying drawings and the following embodiments of the present invention is further illustrated drawings in which:

[0012]图1为本发明实施例贴壁性细胞的三维培养的装置结构示意图。 [0012] FIG. 1 is a schematic configuration example of apparatus adherent cells cultured in a three-dimensional embodiment of the present invention.

具体实施方式 Detailed ways

[0013] 为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。 [0013] To make the objectives, technical solutions and advantages of the present invention will become more apparent hereinafter in conjunction with the accompanying drawings and embodiments of the present invention will be further described in detail. 应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。 It should be understood that the specific embodiments described herein are only intended to illustrate the present invention and are not intended to limit the present invention.

[0014] 本发明实例提出一种贴壁性细胞的三维培养方法,方法的步骤和实施过程包括: [0014] Examples of the present invention proposes a wall of the three-dimensional culture cell attachment process, process steps and the implementation process comprising:

[0015] S10,获取具有细胞亲和性、能用于细胞贴壁性培养的磁珠; [0015] S10, obtaining cells having affinity bead can be used to culture adherent cells;

[0016] S20,以步骤S10的磁珠作为共培养介质与待培养细胞于磁场中进行培养。 [0016] S20, the step S10 as the beads to be co-culture medium cultured cells cultured in a magnetic field.

[0017] 本案中采用上述磁珠作为细胞共培养介质,相比葡聚糖或合成聚合物的微珠培养介质,细胞培养的过程中可以根据各培养时期所要求的空间、密度等条件,通过控制改变磁场的场强(场强是矢量,包括有大小和方向)即可使磁珠的分布产生变化,从而可以根据细胞生长的阶段要求改变培养环境。 [0017] In case the above co-culture medium as a cell beads, beads of dextran or synthetic polymers as compared to culture media, cell culture process may be according to the required space of each culture period, density and other conditions, by changing the magnetic field strength control (field strength vector, including magnitude and direction) can make a difference in the distribution of the magnetic beads, thereby changing the culture environment may be required depending on the stage of cell growth. 而且,通过外加磁场来使磁珠进行运动,可以替代机械搅拌棒、气升式等搅拌使培养液和细胞流动,降低了机械冲击和剪切应力对细胞的伤害。 Further, by applying a magnetic field to make the movement of magnetic beads, can replace mechanical stirrer, airlift culture solution and the like is stirred cell flow, reduces the mechanical impact and shear stress damage to the cells.

[0018] 在上述磁珠培养的实施过程中,磁珠本身要能利于贴壁性细胞的贴壁,而普通的铁、钴材质的磁珠其表面亲和性较差,不利于细胞的贴壁且易被培养液和细胞代谢物腐蚀,所以步骤S10中细胞贴附性的磁珠,可以采用葡聚糖、合成聚合物、亲和树脂等包覆层包覆的复合磁珠,内部被包覆的磁芯用于提供磁性;其表面的包覆层一方面用于细胞的亲和贴附、另一方面可以包覆铁、钴磁芯不被培养液腐蚀。 [0018] In the above-described embodiment the process of the cultured beads, magnetic beads attached itself to be able to facilitate adherence of the cells walls, and ordinary iron, cobalt affinity for a surface material of poor magnetic beads, is not conducive to cell paste wall and easily broth metabolites and cell corrosion, so the step S10 adherent cell beads, dextran beads can be composite, synthetic polymers, and other affinity resin coating layer coated, internally for providing a magnetic core coated; coating layer on the surface thereof an aspect affinity for cell attachment, may be coated on the other hand, iron, cobalt core broth is not corrosive.

[0019] 并且在磁珠的采用过程中,磁珠本身是作为共培养介质,无细胞壁的动物细胞只有贴附在固体基质表面才能增殖,故细胞在微载体表面的贴附是进一步铺展和生长的关键;增大单位体积内表面积(S/F)对细胞的生长非常有利,但是粒径过小时磁珠相对所受的磁场力不利于控制;所以在培养中优选采用粒径200〜500 μπι的磁珠。 [0019] and, in the process using magnetic beads, bead itself as a co-culture medium without cell walls of animal cells only attached to the surface of a solid substrate proliferation, cell attachment so that the surface of the microcarrier further spreading and growth key; increasing the surface area per unit volume (S / F) is very beneficial to cell growth, but the particle size is too small bead suffered opposing magnetic force against a control; and is preferably a particle diameter 200~500 μπι in culture magnetic beads.

[0020] 由于贴壁性的动物细胞,都无细胞壁,对剪切力敏感、极易死亡,因而最好的方式是不用加机械搅拌棒来增加接触概率。 [0020] Since the walls of an animal cell paste, have no cell wall, sensitive to shear forces, easy to death, so the best way is not to increase mechanical stirring rod to increase the contact probability. 本案中的外加磁场一般都是用磁铁构建、或者是交流电产生的交变磁场,通过移动磁铁的距离或者改变交变电流的大小,使磁珠受到的磁场力改变之后产生运动,从而代替搅拌方式增加细胞接触概率。 Magnetic field in this case are generally constructed with a magnet, an alternating current or an alternating magnetic field generated by the alternating current or changing the distance of movement of the magnet size, the changes to the magnetic force generated by magnetic beads after the movement, thereby replacing agitation increase the probability of cell contact.

[0021] 并且进一步在实施的过程中,现有通常采用的微载体是同一密度材质的,所以悬浮在培养液中的统一高度位置;相比本案中尽量采用至少3种不同密度的磁珠进行(密度不同可以通过采用不同磁芯材质、或者不同的磁芯与包覆层厚度比例实现),因为不同密度重量的磁珠一方面在培养液中悬浮于不同的高度,不同密度和体积的磁珠因为自身重力、浮力、磁力不同而处于不同的高度;这样磁珠分布不会全部聚集在同一高度,会呈现密度差,这样有利于细胞的培养;因为细胞在不同的阶段上,需要的这些环境是不同的,接种之后磁珠密度聚集比较多的区域,可以直接更利于早期的生长。 [0021] and further embodiments of the process, generally employed conventional microcarriers same density material, it was suspended in a uniform height position in the culture medium; for this case as far as possible as compared to at least three different densities of magnetic beads (different densities by using different core materials, or various core and cladding layers to achieve a thickness ratio), since the weight of the different densities of the beads are suspended in an aspect different heights in the culture, different magnetic density and volume because these cells in different stages, needs; different beads because of its own gravity, buoyancy, magnetic force at different heights; so that the beads are not distributed all gathered at the same height, will show the difference in density, it is a good cultured cells environment is different, the density of the beads after inoculation more aggregation regions, more conducive to direct the growth of early. 在细胞进入中期之后,细胞慢慢移动或者分散至磁珠密度聚集较低的区域进行生长,并且通过改变外加的磁场,可以调整磁珠悬浮的位置,改变磁珠的密集度,可以分别满足细胞在不同阶段生长环境的要求。 After the cells into the medium-term, slowly moving the cells to magnetic beads or dispersed low density aggregate growth region, and by changing the applied magnetic field, can adjust the position of suspension beads, beads of density change can meet the cells were requirements at different stages of growth environment.

[0022] 进一步,在上述磁珠等等全部确定之后,步骤S20将细胞于磁场中进行培养,磁场的条件控制可以根据所需磁珠的位置和聚集程度进行选择和变化。 [0022] Further, after the determination of all the beads and the like, step S20 cells were cultured in a magnetic field, the magnetic field conditions control can be selected and changed according to the position and degree of aggregation of the desired beads.

[0023] 并且在本案中最优选的方式,磁场优选采用匀强磁场,并且将匀强磁场的方向设置为与磁珠重力方向相反的竖直向上方向,那么这样磁珠在培养液中进行培养时所受的力为始终竖直向下的重力、与重力方向相反的浮力、以及与重力方向相反的磁力。 [0023] and in the most preferred case, the uniform magnetic field magnetic field is preferably employed, and the direction of the uniform magnetic field is disposed opposite to the magnetic beads to the direction of gravity vertically upward direction, so that the beads are cultured in a culture broth always when the force on the vertically downward gravity, buoyancy opposite to the direction of gravity, and magnetic forces opposite to the direction of gravity. 由于其本身是具有铁、钴等磁芯的磁珠,密度上肯定远大于培养液的密度,所以浮力肯定远小于重力,然后通过这一方向上外加的磁力即可作为补充使磁珠能在竖直方向上受力达到平衡而悬浮在培养液中。 Since the bead itself is an iron, cobalt core, certainly much greater than the density of the culture liquid density, buoyancy certainly much less than the weight, then this direction by applying magnetic force to the magnetic beads can be supplemented in the shaft a linear direction of the force equilibrium suspended in the culture medium. 并且,进一步在调整磁场强度大小时,磁珠的运动都是在竖直方向做上下运动,不会出现多向的杂乱碰撞,避免对细胞造成冲击损伤。 And, further in adjusting the size of the magnetic field strength, magnetic motion is up and down movement in the vertical direction, the collision does not appear cluttered and more to avoid impact damage to cells.

[0024] 当然采用其他类型的磁场也可以能满足在内部使磁珠在某一位置平衡,但是这种类型在实施中可能相比不太方便控制磁珠的震动轨迹,可能会比较容易发生相互碰撞。 [0024] Of course, other types of magnetic field may be able to meet in the interior of the magnetic beads at a equilibrium position, but this type of embodiment may be compared to the less easy to control the orbit of vibration of beads, it may be more likely to occur with each other collision. 而且采用上述匀强磁场比较好实施,采用与两块平行板电容器接通直流电之后即可在平行板之间产生上述匀强磁场;非常利于实现本案的上述目的和效果。 And uniform magnetic field using the above-described embodiment is better used to generating the uniform magnetic field between two parallel plates of a parallel plate capacitor after the DC motor is turned on; very conducive to achieving the above object and effect of the case.

[0025] 在上述细胞培养过程完成之后,首先排干培养液,至少用缓冲液漂洗1遍,然后加入相应的酶进行贴壁消化,之后解离即可收集细胞及其产品。 [0025] After completion of the above-described cell culture process, culture medium is first drained, rinsed with buffer at least one pass, then adding the appropriate enzyme digestion Adherent, after dissociation of the cells and their products can be collected.

[0026] 基于本发明的上述采用磁珠进行三维细胞培养的方法,本发明进一步还提出一种实现上述方法的贴壁性细胞的三维培养装置,装置结构可以进一步参见附图1所示,包括圆台状的培养腔10和磁场发生器20 ;其中,磁场发生器20用于向培养腔10提供磁场,最终使培养腔10位于磁场发生器20产生的磁场中。 [0026] The method based on the three-dimensional cell culture using the beads of the present invention, the present invention further proposes a three-dimensional cell culture of adherent apparatus for realizing the method, the device structure may further see Fig. 1, comprising truncated cone-shaped magnetic field generator 10 and the culture chamber 20; wherein the magnetic field generator 20 for providing a magnetic field to the culture chamber 10, and finally to the culture chamber 10 is positioned a magnetic field generator 20 is generated.

[0027] 磁场发生器20可以采用比较通畅的工频磁场发生器、或者是一些磁铁等永磁性物体均可,并且调整其强度至能使磁珠在添加有培养液的培养腔10中能悬浮即可。 [0027] The magnetic field generator 20 may employ the relatively smooth frequency magnetic field generator, or some of the objects can be a permanent magnet or the like, and can adjust the intensity of the magnetic beads is added to the culture medium of the cavity 10 can be suspended It can be.

[0028] 同时,装置中的培养腔10采用圆台形的性状设计,并且圆台形的性状为从底部向顶部的方向截面直径逐渐增大。 [0028] Meanwhile, in the culture chamber unit 10 employs truncated cone design traits, and traits of truncated cone cross-sectional diameter from the bottom to top direction gradually increases. 这一形状相比普通的圆柱形的细胞培养腔,在细胞接种培养的早期,可以控制磁场发生器20发出的磁场的强度在相对较小的程度,使磁珠平衡悬浮在培养腔10的下方空间内,这样磁珠之间的密集度比较高一些,适合于早期数量较少的细胞粘附和扩增;当细胞开始扩增并且贴壁牢固之后,调整加大电磁场的强度;在磁场的作用下,磁珠会携带这干细胞或者肿瘤细胞漂浮至更高的位置。 The cylindrical shape compared to ordinary cell culture chamber, cells were seeded in early culture, can control the intensity of the magnetic field emitted by the magnetic field generator 20 in a relatively small extent, the magnetic beads were suspended in equilibrium under the culture chamber 10 the space, between such beads intensity relatively higher for a smaller number of early cell adhesion and amplification; when cells began after amplification and firmly adherent, adjusted to increase the strength of the electromagnetic field; in the magnetic field under the action of magnetic beads will carry the tumor cells, or stem cells to a higher floating position. 根据细胞数量和培养需求,来调整磁珠在容器中悬浮的高度,以确保细胞不会粘附到培养容器底部,而是悬浮在液体中的合适位置培养。 The cell culture and the number of demand, to adjust the magnetic beads suspended in the vessel height, in order to ensure that the cells do not adhere to the bottom of the culture vessel culture place, but suspended in the liquid. 这样可以进一步缩短细胞的生长配置的周期,而且可以降低细胞培养过程中造成的细胞损伤,相比通常的微载体培养方式效果更好一些。 This can further shorten the cell growth cycle configuration, and can reduce cell damage caused by cell culture, compared to the usual manner microcarrier culture better effect.

[0029] 因此,基于本案的上述匀强磁场的优选实施效果,上述磁场发生器20优选采用平行板电容器实现,附图1中即为这一方式,因为平行板电容器的两个平板分别接通在直流电的正负极之后,两块平板之间会产生匀强电场、及与电场垂直的匀强磁场。 [0029] Accordingly, preferred embodiments of the effects of the uniform magnetic field based on the case, the magnetic field generator 20 is preferably employed to achieve a parallel plate capacitor, namely the drawings Embodiment 1, since the two flat parallel plate capacitors are switched after the positive and negative direct current, generated between two flat uniform electric field and uniform magnetic field perpendicular to the electric field. 所以,基于本案的目的和效果,将平行板电容器的两块平板沿竖直方向平行设置,即可产生竖直方向的磁场。 Therefore, the purpose and effect of the case, the two plates in the vertical direction parallel plate capacitor are arranged in parallel to the vertical direction magnetic field is generated. 然后通过直流电的正负极方向和大小的即可改变磁场的大小和方向(向上或者是向下),即可构建本案的上述方法实施装置。 Then to change the size and direction of the magnetic field by the positive and negative direction and magnitude of a direct current (up or down), the case of the method described above can be constructed embodiment apparatus.

[0030] 以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包括在本发明的保护范围之内。 [0030] The foregoing is only preferred embodiments of the present invention but are not intended to limit the present invention, any modifications within the spirit and principle of the present invention, equivalent substitutions and improvements should be included in the present within the scope of the invention.

Claims (4)

1.一种贴壁性细胞的三维培养方法,其特征在于,包括: 获取细胞贴壁性的磁珠; 以所述磁珠作为共培养介质与待培养细胞于磁场中进行培养。 1. A three-dimensional cell culture method paste wall, characterized in that, comprising: acquiring magnetic beads attached to the wall of the cell; beads to the co-culture medium as a magnetic field to be cultured in the cultured cells.
2.如权利要求1所述的贴壁性细胞的三维培养方法,其特征在于,所述磁场为匀强磁场,且磁场的方向为与磁珠重力方向相反。 2. The three-dimensional culture of adherent cells according to claim 1, characterized in that the magnetic field is a uniform magnetic field, and the magnetic field direction is opposite to the direction of gravity and magnetic beads.
3.如权利要求1或2所述的贴壁性细胞的三维培养方法,其特征在于,所述磁珠由至少三种不同密度的磁珠组成。 Three-dimensional culture of adherent cells or claim 12, wherein said beads comprised of at least three different densities bead composition.
4.如权利要求1或2所述的贴壁性细胞的三维培养方法,其特征在于,所述磁珠的粒径为200 〜500 μπι。 4. The three-dimensional culture of adherent cells of claim 1 or claim 2, wherein the bead diameter is 200 ~500 μπι.
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US20020090741A1 (en) * 2001-01-08 2002-07-11 Jurgensen Stewart Russell Method of separating cells from a sample
CN104694474A (en) * 2015-03-31 2015-06-10 南京新诺丹生物技术有限公司 Cell culture method

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US20020090741A1 (en) * 2001-01-08 2002-07-11 Jurgensen Stewart Russell Method of separating cells from a sample
CN104694474A (en) * 2015-03-31 2015-06-10 南京新诺丹生物技术有限公司 Cell culture method

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105671029A (en) * 2016-03-08 2016-06-15 万香波 Establishment method of three-dimensional cell model

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