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March 2020

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A Method to Efficiently Cryopreserve Mammalian Cells on Paper Platforms
一种纸质平台高效冷冻保存哺乳动物细胞的方法   

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Abstract

This protocol describes a simple method to cryopreserve mammalian cells within filter papers as an alternative to conventional slow-freezing approach. The method involves treating paper fibers with fibronectin, using low concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), and slow freezing cells to -80 °C at a 1 °C min-1 rate. In our method, the biocompatibility, large surface area, 3D porosity and fiber flexibility of the paper, in combination with the fibronectin treatment, yield recovery of cells comparable to conventional approaches, with no additional fine-tuning to freezing and thawing procedures. We expect that the paper-based cryopreservation method will bring several advantages to the field of preserving mammalian cells, including accommodation of a higher number of cells within a unit volume and no cell loss after release. The method requires a minimal storage space, where paper platforms with large areas can be rolled and/or folded and stored in stocks, and allows for efficient transportation/distribution of cells in an on-demand manner. Moreover, an additional feature of this method includes the formation and cryopreservation of cellular spheroids and 3D cell cultures.

Keywords: Paper (纸), Mammalian cells (哺乳动物细胞), Cryopreservation (低温贮藏), Storage (存贮), Retrieve (恢复), Release (释放)

Background

Successful preservation, long-term storage, maintenance and distribution of mammalian cells are important research areas that are still under intense scientific investigation. In particular, the timely and steady supply of frozen cells is pertinent in tissue engineering research such as cell culture, drug development and testing, and regenerative and biotherapeutic medicine.

Current conventional cell cryopreservation protocols include slow and rapid freezing and vitrification (Pegg, 2002; Baust et al., 2009). In these approaches, cryoprotectant agents at various concentrations are added to the cell suspension, followed by cooling down the medium at temperature rates as low as 1 °C min-1 (slow freezing) to as high as 120 °C min-1 (rapid freezing), or by placing samples directly in -195 °C liquid nitrogen tanks (vitrification). As a result, with the protective role of cryoprotectants, the cell damage or death is minimized during freezing (Karlsson and Toner, 1996; Asghar et al., 2014; Jang et al., 2017). However, these approaches possess limitations in terms of requiring large storage spaces to house the cells in thousands to millions of small cryotubes and cryobags (Heidemann et al., 2010; Massie et al., 2014). The distribution of cells then faces challenges in terms of loss, misidentification, and logistics management (Tomlinson, 2005).

Recent approaches described the use of various engineered porous scaffolds for cryopreservation of cell tissue constructs. Examples include the use of corn starch-polycaprolactone fiber meshes (Costa et al., 2012), electrospun-polyurethane nanofiber sheets (Batnyam et al., 2017), alginate-gelatin cryogel sponges (Katsen-Globa et al., 2014), and reticulated polyvinyl formal resins (Miyoshi et al., 2010). The results of these studies proved that the biocompatibility, mechanical support, and 3D porosity of scaffolds provide a suitable balance in creating a protective microenvironment for the cells during their cryopreservation. However, with these scaffolds, substrates need to be repeatedly manufactured (i.e., engineered) for their use in relevance to cryopreservation of cells, which brings a heavy dose of hurdle to the field.

Remarkably, paper-based platforms have emerged as an attractive alternative for tissue engineering development, and especially for 3D cell culture. With added features such as cost-effectiveness and tunable fiber surface characteristics, these ready-to-use scaffolds offer remarkable applicability for large-scale, multilayer biological testing (Derda et al., 2009; Mosadegh et al., 2014). As a result, various cellular applications utilizing paper platforms have been intensively investigated (Ng et al., 2017; Wu et al., 2018; Rosqvist et al., 2020). Yet, despite its outstanding potential as a substrate for 3D cell culture and molecular sampling, paper platform has never been utilized to its full potential in directly cryopreserving cells, until our recent work (Alnemari et al., 2020). Instead, it was used as a vitrification container (2D paper substrate) to enhance cryopreservation of mouse embryos (Paul et al., 2018), bovine oocytes (Kim et al., 2012), and bovine blastocysts (Lee et al., 2013). FTA cards, on the other hand, were used for the collection, storage (at room temperature or at +4 °C, -20 °C and -80 °C), transportation, and molecular analysis of nucleic acids (Santos, 2018).

In this protocol, we give step-by-step explanations (Figure 1) on how to 3D preserve and release mammalian cells using our developed paper-based cryopreservation method (Alnemari et al., 2020). The technique starts with cutting the filter paper into small strips (e.g., 3 × 3 cm2). Then, paper fibers are treated with fibronectin to enhance the post-thawing cell release. This is trailed by suspending cells in serum medium containing low concentration dimethyl sulfoxide (DMSO). Following, cell suspension is pipetted onto fibronectin-treated papers. Immediately after cells penetrate within the paper’s 3D porous matrix, papers are rolled and placed in standard cryotubes, slow freezed to -80 °C at a 1 °C min-1 rate, and kept in -195 °C liquid nitrogen for long-term storage. The cells can then be thawed and, depending on the need, either released from the paper to expand as typical 2D culture in flasks or kept inside the paper to grow as 3D cultures and spheroids.

In the developed method, cells are ubiquitous in the 3D porous environment of the paper, where paper fibers provide a natural protective and supportive environment during their cryopreservation. As a result, after their freeze, thawed cells are efficiently released from paper with high viability rates by gently shaking the paper. Here, any paper type, with pores suitable for cells to penetrate within, can be used by simply optimizing the fibronectin concentration for the effective release of cells. The paper also provides a versatile environment for the remaining cells within the paper to grow as aggregates (spheroids) and, as well, successfully enables the formation (by using Matrigel matrix) and cryopreservation of 3D cell cultures (Alnemari et al., 2020). The paper-based cryopreservation offers space-saving and efficient cell transportation/distribution solutions in a cost-effective, fast and easy-to-manage manner, since large paper sheets can be rolled and/or folded to fit standard cryotubes (or other contianers) and stored in stocks and cut into small pieces without the need to thaw the entire platform.


Figure 1. Paper-based cell cryopreservation method. A. An outline of stages involved in the paper-based cryopreservation method. After the thaw, cells can be either released from paper by gentle shaking and expand as 2D culture in flasks or 3D cultured in vitro as needed. B. Micrographs visualize the way a 3 × 3 cm2 paper strip is treated and placed in a cryotube after rolling.

Materials and Reagents

The materials and reagents presented below are for paper-based cryopreservation of cervical HeLa cell lines. For cryopreservation of breast MCF-7, prostate PC3 and lymphocyte JKT cell lines, see the section “Notes” for specific details.

Required

  1. Sterile 10 ml serological pipettes (Costar Stripette, catalog number: 4488 )
  2. Sterile 15 ml centrifuge tubes (ThermoFisher, catalog number: 339650 )
  3. Sterile 1-20 μl, 200 μl, 1,000 μl pipette tips (ThermoFisher, catalog numbers: 10380792 , 10619331 , 10390792 )
  4. Sterile T75 tissue culture flasks (ThermoFisher, catalog number: 156499 )
  5. Sterile 1 ml cryogenic tubes (cryotubes) (Sigma-Aldrich, catalog number: CLS430487 )
  6. Cryo-safe vial storage boxes (cryoboxes) (Sigma-Aldrich, catalog number: Z756776 )
  7. Mr. Frosty freezing container (ThermoFisher, catalog number: 5100-0001 )
  8. Sterile 35 mm x 10 mm Petri dishes (Corning, catalog number: 430165 )
  9. Sterile 60 mm x 10 mm Petri dishes (Falcon, catalog number: 351007 )
  10. Whatman Grade 114 cellulose filter papers (Sigma-Aldrich, catalog number: 1114-185 )
  11. Warm water (37 °C)
  12. Liquid nitrogen (-196 °C)
  13. Dulbecco’s phosphate buffered saline (DPBS)-10x (Sigma-Aldrich, catalog number: 59331C )
  14. Fibronectin human plasma (Sigma-Aldrich, catalog number: F0895 )
  15. Human cervical HeLa cancer cell line (ATCC, catalog numbers: CCL-2 )
  16. Dulbecco's modified Eagle’s medium (DMEM) (Gibco, catalog number: 11965092 )
  17. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
  18. Penicillin-streptomycin (Pen-Strep) solution (Sigma-Aldrich, catalog number: P4333 )
  19. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  20. TrypLE express enzyme (Gibco, catalog number: 12604021 )
  21. Corning Matrigel matrix (Sigma-Aldrich, catalog number: DLW356231 )

Optional
  1. Cell counting chamber slides (ThermoFisher, catalog number: C10228 )
  2. Trypan blue exclusion assay (Optional, Sigma-Aldrich, catalog number: T6146 )
  3. Invitrogen Live/Dead assay kit for mammalian cells, containing green calcein-AM and red ethidium homodimer-1 fluorescent dyes (ThermoFisher, catalog number: L3224 )
  4. Complete Roswell Park Memorial Institute (RPMI) medium (Gibco, catalog number: 11875093 )

Equipment

Required

  1. All-purpose scissors with size of 18 cm
  2. Ruler of length 15 cm
  3. High precision tweezers (Dumont, catalog number: 5627 )
  4. Timer (Sunnex, catalog number: 360594 )
  5. Microbalance (Mettler Toledo, catalog number: ME54 )
  6. Autoclave (Runyes, catalog number: SEA 18L-DIG-USB )
  7. Centrifuge (Eppendorf, catalog number: 5810 )
  8. Laboratory water bath (Witeg, catalog number: WITEG 20002 )
  9. Humidifying cell incubator (New Brunswick, catalog number: Galaxy 48R )
  10. Class II laminar air flow hood (NuAire, catalog number: NU-437S )
  11. -80 °C freezer (Arctiko, catalog number: ULUF 65 )
  12. Liquid nitrogen tank (Arpege, catalog number: 40 )

Optional
  1. Countess II FL automated cell counter (ThermoFisher, catalog number: AMQAF1000 )
  2. Olympus FV1000 inverted confocal microscope (Olympus America)

Software

  1. (Optional) Imaris 9.2 image analysis software (Oxford Instruments)

Procedure

  1. Preparation of paper (see Video 1)
    1. Autoclave the paper and cut it, using sterile scissors, into ~3 x 3 cm2 strips under the hood (Papers can be cut to other sizes and shapes as well, provided that the pipetted cell suspension saturates the paper. See Notes for details).
    2. Place the paper strips in sterile 60 mm x 10 mm Petri dishes (one strip per dish) and pipette 100 µl DPBS containing 10 µg/ml fibronectin concentration under the hood. After adding the fibronectin solution, incubate the paper fibers for 20 min at room temperature. To avoid water evaporation, make a humidifying chamber by using wet tissues inside Petri dishes and cover their lid (see Notes for the evaporation rate).
    3. Remove the excess fibronectin by rinsing the paper strips twice with 100 µl DPBS for about 5 s.
    4. Keep the fibronectin-treated paper strips in the humidifying chamber until the cells are prepared and loaded.

      Video 1. Steps involved in preparation of paper strips for use in cryopreserving mammalian cells. Paper strips of any type, size and shape can be used provided that the paper pores are suitable for cells to penetrate within and the fibronectin is saturating the paper.

  2. Preparation of cells
    1. Culture ~106 cells in T-75 flasks using 10 ml complete DMEM (see Recipes) and place them in a humidifying incubator at 37 °C and 5% CO2.
    2. Passage cells for a maximum of 15 to 24 passages.
    3. Dissociate the cells from the flasks when they are ~80% confluent using 2 ml TrypLE for 5 min. Transfer the cells to 15 ml centrifuge tubes to centrifuge at 300 x g for 5 min at 37 °C.
    4. Resuspend the cell pellet (~107 cells) in 1 ml room temperature DPBS and apply trypan blue exclusion assay to count the number of live and dead cells using cell counting chamber slides and automated cell counter.
    5. Centrifuge cells at 300 x g for 5 min at 37 °C and resuspend the cell pellet (~107 cells) in 300 µl DMEM-based freezing medium (see Recipes).

  3. Cryopreservation of cells (see Video 2)
    1. Transfer fibronectin-coated paper strips to sterile 35 mm x 10 mm Petri dishes (one strip per dish) and pipette onto them 300 µL of cell suspensions (~107 cells/ml per cm2, see Notes).
    2. Roll the paper strips with tweezers within 1 min to prevent evaporation of cell suspension and place them immediately in cryotubes. Then, place the cryotubes in Mr. Frosty freezing container. To control the rate of freezing, fill the container with 100% isopropyl alcohol to the indicated line.
    3. Slow freeze the cells in Mr. Frosty container for overnight to -80 °C at a -1 °C min-1 rate.
    4. Transfer the cells to cryoboxes for long-term storage in liquid nitrogen at -196 °C.

      Video 2. Cell loading into paper strips. Roll the paper strips and place them in standard cryotubes within one minute to prevent evaporation of the suspension medium.

  4. Thawing cells on-demand
    1. Take the cryotubes from liquid nitrogen and thaw them in water bath at 37 °C for 30 s.
    2. Cells can be cryopreserved in larger sheets. In this case, cut a small portion of the sheet using sterilized scissors, followed by thaw. Place the unused sheet back in liquid nitrogen. Verify the cell viability on sheets after each sample retrieval cycle.

  5. Releasing cells from paper
    1. Remove the cell-loaded paper strips from the cryotubes under the hood and place them in 15 ml centrifuge tubes containing 10 ml of complete DMEM.
    2. Manually shake centrifuge tubes for about 20 s to release the cells from paper.
    3. Remove the paper strips from the tubes using sterile tweezers.
    4. Centrifuge the cell suspension in the tubes at 300 x g for 5 min at 37 °C to pellet the cells.
    5. Wash the cell pellet three times with 1 ml room temperature DPBS.
    6. Resuspend the cells in complete DMEM in T75 flasks and place the flasks in humidifying incubator at 37 °C and 5% CO2 to expand them as 2D culture (Figure 2).


      Figure 2. Confocal microscope images show cells expanded as 2D culture after paper-based and conventional (control) cryopreservation. Following their proliferation for 3 days, the spread of actin (red) and tubulin (green) and the presence of nucleus (blue) confirmed no morphological abnormalities or differences between paper-retrieved and freshly cultured HeLa cells.

    7. (Optional) Apply trypan blue exclusion assay to directly count the number of released live and dead cells using cell counting chamber slides and automated cell counter.
    8. (Optional) Utilize the cells that still remain on the paper (Figure 3) for further in vitro 3D culture growth and spheroid formations (Alnemari et al., 2020) or any other cellular application. This is achieved by placing the paper, with remaining cells, in sterile Petri dishes and allowing cells to 3D expand inside humidifying incubator at 37 °C and 5% CO2. Note that this step has been extended in our recent study (Alnemari et al., 2018) to form arrays of 3D cell cultures on paper platforms, and currently we are advancing its applicability to cryopreserving these arrays.

  6. Cryopreservation of 3D cell cultures
    1. Add cells (~107 cells/ml concentration) to 100% Matrigel at 4 °C (placed on ice) and subsequently pipette the cell suspensions in Matrigel onto the autoclaved paper strips placed in sterile Petri dishes (~107 cells/ml per cm2).
    2. Leave paper strips for 10 min at room temperature for the Matrigel to solidify.
    3. Submerge paper strips in complete DMEM for 7 days in humidifying incubator at 37 °C and 5% CO2 to allow the proliferation of cells within the paper.
    4. Roll the paper strips and place them in standard cryotubes containing DMEM-based freezing medium. Place the cryotubes in cryoboxes.
    5. Slow freeze the proliferated cells for overnight to -80 °C at a 1 °C min-1 rate.
    6. Transfer the cells to liquid nitrogen at -196 °C for long-term storage.


      Figure 3. Confocal microscope images show the viable cell release from paper after thaw. Following their load onto 10 µg/ml fibronectin-treated papers and subsequent freeze and thaw, the relative distribution of live (green) and dead (red) HeLa cells within the paper platform visually demonstrate the effective release of viable cells from paper.

  7. Thawing and providing 3D cell cultures on-demand
    1. Take the cryotubes from liquid nitrogen and thaw them in water bath at 37 °C for 30 s.
    2. In case the cell culture is cryopreserved in larger sheets; cut a small portion of the sheet, followed by thaw. Place the unused sheet back in liquid nitrogen.
    3. Submerge the paper strips with remaining cells in complete DMEM in sterile Petri dishes for additional cell proliferation in humidifying incubator at 37 °C and 5% CO2.
    4. (Optional) Use Live/Dead assay to visually assess (as control) the distribution of live/dead cells within the papers (Figure 4). This is a useful step especially when working with 3D cell cultures (see Recipes for procedure details).

Data analysis

The Live/Dead analysis was carried by imaging cells with Olympus FV1000 inverted confocal laser scanning microscope (Olympus Corporation) using green (488 nm), and red (612 nm) excitation wavelengths and post-processing images with Imaris software. 10× air objective lens was used to image up to 160 µm deep in paper. The z-stack imaging was performed in 5 µm increments and their 3D projections were created using Imaris software.


Figure 4. Confocal microscope image shows the survival of cells in paper-based 3D culture after thaw. Following 7 days of 3D paper-based HeLa cell culture, freezing for overnight to -80 °C at a 1 °C min-1 rate, and finally thawing; experiments showed integrity and survival of viable cells.

Notes

  1. Breast MCF-7 cancer cell line, prostate PC3 cancer cell line, and lymphocyte JKT cell line (ATCC, catalog numbers: CRL-3435, CRL-1435, and TIB-152, respectively) can also be used for paper-based cryopreservation. See Alnemari et al. (2020) for comparison. Below are details of procedures for preparing the cells. The rest of the cryopreservation and thawing procedures are the same when HeLa cells are used.
    1. Use complete DMEM for MCF-7 cells and complete RPMI medium for PC3 and JKT cells, both supplemented with 10% (vol/vol) FBS and 1% (vol/vol) Pen-Strep.
    2. Dissociate PC3 and MCF-7 cells from the flasks when they are ~80% confluent using TrypLE and centrifuge at 300 x g for 5 min. Collect suspended JKT cells and centrifuge at 200 x g for 7 min.
    3. Resuspend the cell pellets (~107 cells) in complete freezing medium with 10% (vol/vol) DMSO for PC3 and MCF-7 cells, and with 5% (vol/vol) DMSO for JKT cells.
  2. The change in the water mass within the paper at room temperature (water evaporation rate) is 0.002 g min-1 per 9 cm2 paper strips.
  3. 300 µl cell suspension (107 cells/ml concentration) is just enough to saturate the 3 × 3 × 0.02 cm3 paper volume with cells.

Recipes

  1. Complete medium
    To prepare complete cell culture medium for HeLa cells, supplement the DMEM with 10% (vol/vol) FBS and 1% (vol/vol) Pen-Strep
  2. Freezing medium
    To prepare freezing medium for HeLa cells, add 10% (vol/vol) DMSO to complete DMEM
  3. Live/Dead assay
    1. Dilute 2.5 µl of calcein-AM and 10 µl ethidium homodimer-1 in 5 ml room temperature DPBS
    2. Pipette 100 µl of working solutions onto paper stripes and incubate for 30 min in the dark
    3. Place the paper on coverslip and seal it with another coverslip using mounting medium
    4. Observe the cells under microscope for the integrity, survival, and viability

Acknowledgments

The work was financially supported by NYU Abu Dhabi (NYUAD). We acknowledge the technical support from Core Technology Platforms at NYU Abu Dhabi. This protocol was revised from the procedures described in Alnemari et al. (2020).

Competing interests

The authors declare no competing interest.

References

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简介

[摘要] 该协议描述了一种简单的方法,可在滤纸中冷冻保存哺乳动物细胞,以替代常规的慢速冷冻方法。该方法包括使用纤连蛋白处理纸纤维,使用低浓度的冷冻保护剂二甲基亚砜(DMSO),然后以1°C min -1的速率将细胞缓慢冷冻至-80°C 。在我们的方法中,纸的生物相容性,大表面积,3D孔隙率和纤维柔韧性与纤连蛋白处理相结合,可产生与传统方法相当的细胞回收率,而无需对冷冻和解冻程序进行额外的微调。我们期望纸质冷冻保存方法这将为保存哺乳动物细胞领域带来几项优势,包括在单位体积内容纳更多数量的细胞,并且释放后无细胞损失。该方法需要最小的存储空间,在该存储空间中,可以将具有大面积的纸平台卷起和/或折叠并存储在库存中,并允许按需方式有效地运输/分配细胞。此外,该方法的另一个特征包括细胞球体和3D细胞培养物的形成和冷冻保存。


[背景] 哺乳动物细胞的成功保存,长期保存,维护和分配是​​重要的研究领域,目前仍在深入的科学研究中。特别是,冷冻细胞的及时稳定供应与组织工程研究有关,例如细胞培养,药物开发和测试以及再生和生物治疗医学。

当前的常规细胞冷冻保存方案包括缓慢和快速的冷冻和玻璃化(Pegg,2002; Baust 等,2009)。在这些方法中,将各种浓度的冷冻保护剂添加到细胞悬浮液中,然后以低至1°C min -1 (慢速冷冻)至高至120°C min -1 (快速)的温度冷却介质。冷冻),或将样品直接放在-195°C液氮罐中(玻璃化)。结果,由于具有冷冻保护剂的保护作用,在冷冻过程中细胞的损伤或死亡被最小化(Karlsson和Toner,1996; Asghar 等,2014; Jang 等,2017)。然而,这些方法在需要大的存储空间以将细胞容纳在成千上万个小的冷冻管和冷冻袋中方面具有局限性(Heidemann 等,2010; Massie 等,2014)。然后,细胞的分布面临着损失,识别错误和物流管理方面的挑战(Tomlinson,2005 )。

最近的方法描述了使用各种工程多孔支架进行细胞组织构建物的冷冻保存。示例包括使用玉米淀粉-聚己内酯纤维网(Costa 等人,2012),静电纺丝-聚氨酯纳米纤维片(Batnyam 等人,2017),藻酸盐-明胶冷冻凝胶海绵(Katsen-Globa 等人,2014),以及网状聚乙烯醇缩甲醛树脂(Miyoshi et al。,2010)。这些研究的结果证明,支架的生物相容性,机械支持和3D孔隙度在为细胞冷冻保存创造保护性微环境时提供了适当的平衡。然而,对于这些支架,需要针对与细胞的冷冻保存相关的用途重复制造(即,工程化)基质,这给该领域带来了很大的障碍。

值得注意的是,基于纸的平台已成为组织工程开发,尤其是3D细胞培养的有吸引力的替代方案。这些现成的支架具有成本效益和可调节的纤维表面特性等附加功能,为大规模,多层生物测试提供了出色的适用性(Derda 等,2009; Mosadegh 等,2014)。结果,已经广泛地研究了利用纸平台的各种蜂窝应用(Ng 等,2017; Wu 等,2018; Rosqvist 等,2020)。然而,尽管纸平台作为3D细胞培养和分子采样的基质具有巨大潜力,但直到我们最近的工作(Alnemari 等人,2020年)之前,纸平台从未被充分利用来直接冷冻保存细胞。取而代之的是,它被用作玻璃化容器(2D纸质基材)以增强小鼠胚胎(Paul 等人,2018),牛卵母细胞(Kim 等人,2012)和牛胚泡(Lee 等人,2013 )的冷冻保存。)。另一方面,FTA卡用于核酸的收集,存储(在室温或+4°C,-20°C和-80°C下),运输和分子分析(Santos,2018)。

在此协议中,我们给出了有关如何使用我们开发的基于纸张的冷冻保存方法(Alnemari 等,2020)对3D保存和释放哺乳动物细胞进行分步说明(图1 )。该技术始于切割滤纸切成小条带(例如,3 ×3cm的2 )。然后,用纤连蛋白处理纸纤维以增强解冻后细胞的释放。这是通过将细胞悬浮在含有低浓度二甲基亚砜(DMSO)的血清培养基中来进行的。随后,将细胞悬浮液吸移到纤连蛋白处理的纸上。细胞渗入纸张的3D多孔基质后,立即将其卷起并放入标准冷冻管中,以1°C min -1的速率缓慢冷冻至-80°C ,并长期保持在-195°C液氮中储存。然后可以将细胞融化,并根据需要从纸中释放出来以在烧瓶中以典型的2D培养物形式进行扩展,或者保存在纸中以以3D培养物和类球体的形式生长。

在开发的方法中,细胞在纸张的3D多孔环境中无处不在,其中纸张纤维在冷冻保存期间提供了自然的保护和支持环境。结果,融化后的细胞通过轻轻摇动纸,以高存活率有效地从纸上释放。在此,可以通过简单地优化纤连蛋白浓度以有效释放细胞来使用具有适合细胞穿透的孔的任何类型的纸张。该论文还为论文中的其余细胞以聚集体(球状体)的生长提供了通用的环境,并且还成功地实现了3D细胞培养物的形成(通过使用Matrigel基质)和冷冻保存(Alnemari 等人,2020)。。纸基冷冻保存以节省成本,快速且易于管理的方式提供节省空间和有效的细胞运输/分配解决方案,因为可以卷起和/或折叠大纸张以适合标准冷冻管(或其他容器))并储存在库存中并切成小块,无需解冻整个平台。


图1. 纸基细胞冷冻保存方法。一。纸质冷冻保存方法所涉及的阶段概述。解冻后,可以通过轻轻摇动将细胞从纸中释放出来,并在烧瓶中以2D培养物的形式扩增,或者根据需要在体外进行3D培养。乙。显微照片可视化处理3×3 cm 2的纸带并在滚动后将其放置在冷冻管中的方式。

关键字:纸, 哺乳动物细胞, 低温贮藏, 存贮, 恢复, 释放

材料和试剂
以下介绍的材料和试剂用于宫颈HeLa细胞系的纸基冷冻保存。对于乳腺MCF-7,前列腺PC3和淋巴细胞JKT细胞系的冷冻保存,请参阅“注意”部分以了解详细信息。

需要
1. 无菌10米升血清移液管(Costar公司Stripette ,目录号:4488)      
2. 无菌15米升离心管(赛默飞,目录号:339650)      
3. 无菌1-20μ 升,200μ 升,1 ,000μ 升移液管尖端(赛默飞,目录号:10380792,10619331,10390792)      
4. 无菌T75组织培养瓶(ThermoFisher,目录号:156499)      
5. 无菌1米升低温管(冷冻管)(Sigma-Aldrich公司,目录号:CLS430487)      
6. 冷冻安全的样品瓶储存盒(冷冻盒)(Sigma-Aldrich,目录号:Z756776)      
7. 弗罗斯蒂先生冷冻容器(ThermoFisher,目录号:5100-0001)       
8. 35毫米x 10毫米无菌培养皿(Corning,目录号:430165)       
9. 60毫米x 10毫米无菌培养皿(猎鹰,目录号:351007)       
10. Whatman 114级纤维素滤纸(Sigma-Aldrich,目录号:1114-185)   
11. 温水(37°C)   
12. 液氮(-196°C)   
13. Dulbecco磷酸盐缓冲盐水(DPBS)-10x(Sigma-Aldrich,目录号:59331C)   
14. 纤连蛋白人血浆(Sigma-Aldrich,目录号:F0895)   
15. 人宫颈HeLa癌细胞系(ATCC,目录号:CCL-2)   
16. Dulbecco改良的Eagle培养基(DMEM)(Gibco,目录号:11965092)   
17. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)   
18. 青霉素链霉素(Pen-Strep)溶液(Sigma-Aldrich,目录号:P4333)   
19. 二甲基亚砜(DMSO)(西格玛奥德里奇,目录号:D2650 )   
20. TrypLE 表达酶(Gibco,目录号:12604021)   
21. 康宁基质胶基质(Sigma-Aldrich,目录号:DLW356231 )   

可选的
1. 细胞计数室载玻片(ThermoFisher,目录号:C10228)       
2. 锥虫蓝排除试验(可选,Sigma-Aldrich,目录号:T6146 )       
3. 用于哺乳动物细胞的Invitrogen 活/死测定试剂盒,其中包含绿色钙黄绿素-AM和红色乙锭均二聚体-1荧光染料(ThermoFisher,目录号:L3224)       
4. 完整的罗斯威尔公园纪念学院(RPMI )介质(Gibco,目录号:11875093)       

设备
需要
尺寸18 厘米的通用剪刀
尺长15厘米
高精度镊子(杜蒙,目录号:5627)
计时器(Sunnex ,目录号:360594)
微量天平(梅特勒-托利多,目录号:ME54)
高压灭菌器(Runyes ,目录号:SEA 18L-DIG-USB)
离心机(Eppendorf,目录号:5810)
实验室水浴箱(Witeg ,目录号:WITEG 20002)
加湿细胞培养箱(新不伦瑞克省,目录号:Galaxy 48R)
II级层流通风罩(NuAire ,目录号:NU-437S)
-80 °C 冰箱(Arctiko ,目录号:ULUF 65)
液氮罐(Arpege ,目录号:40)
 
可选的
Countess II FL自动细胞计数仪(ThermoFisher,目录号:AMQAF1000)
奥林巴斯FV1000倒置共聚焦显微镜(Olympus America)


软件
(可选)Imaris 9.2图像分析软件(牛津仪器)

程序
PAP的制备ER(小号EE V 记意1 )
高压釜的纸张和切割它,使用无菌剪刀,进入〜3×3厘米2 条罩下(论文可以是切向其他尺寸和形状,以及,提供的移液细胞悬浮液饱和的详细信息参见注释的文件内。) 。
将纸条放入无菌的60 mm x 10 mm 培养皿中(每个培养皿一条),并在通风橱下用移液管吸取100 µl 含10 µg / ml 纤连蛋白的DPBS 。后加入纤连蛋白溶液,将纸纤维在室温下孵育20分钟。为避免水分蒸发,请使用培养皿内部的湿纸巾盖住盖子,形成一个加湿室(蒸发速率请参见注释)。
用100 µl DPBS 冲洗纸条两次约5 s,以除去多余的纤连蛋白。
将经过纤连蛋白处理的纸条放在加湿室中,直到准备好细胞并装入细胞。

视频1. 制备用于冷冻保存哺乳动物细胞的纸条的步骤。可以使用任何类型,大小和形状的纸条,只要纸孔适合细胞穿透并且纤连蛋白使纸饱和即可。
 
制备细胞
使用10 ml 完整的DMEM 在T-75烧瓶中培养约10 6个细胞(请参见食谱),并将其置于37 °C 和5%CO 2 的加湿培养箱中。
传代细胞最多15到24代。
使用约80%的融合液将细胞从烧瓶中解离 2 m l TrypLE,持续5分钟。 将细胞转移至15 ml 离心管中,在37 °C下以300 x g 离心5分钟。
在1 ml 室温DPBS中重悬细胞沉淀(〜10 7个细胞),并使用细胞计数室载玻片和自动细胞计数器进行台盼蓝排除试验,以计算活细胞和死细胞的数量。
离心细胞,在300 ×g下在37 5分钟℃,和重悬细胞沉淀(约10 7 个细胞)我Ñ 300μ 升 基于DMEM冷冻培养基(见配方)。
 
细胞的冷冻保存(小号EE V 记意2 )
转印纤连蛋白涂覆的纸条至无菌35 毫米×10毫米的培养皿中(每个培养皿一个条带)和移液管到他们300μL细胞悬浮液(〜10个7 细胞/米升每cm 2,见注解)。
在1分钟内用镊子将纸条滚动,以防止细胞悬浮液蒸发,并立即将其放入冷冻管中。然后,将冷冻管放入Frosty先生的冷冻容器中。要控制冷冻速度,请在容器中加入100%异丙醇至指示的行。             
将细胞缓慢冷冻于Mr. Frosty容器中,以-1°C min -1的速率过夜冷冻至-80 ° C 。
将细胞转移到冷冻箱中,以便在-196 ° C的液氮中长期保存。
 
V IDEO 2 。细胞装入纸条中。滚动纸条并将其放在标准冷冻管中,在一分钟之内,以防止悬浮介质蒸发。

按需解冻细胞
从液氮中取出冷冻管,并在37 ° C的水浴中融化30秒钟。
可以将细胞冷冻保存在较大的薄片中。在这种情况下,请使用消毒剪刀剪掉一小部分纸,然后解冻。将未使用的纸张放回液氮中。在每个样品取回周期后,在纸上验证细胞活力。
 
从纸上释放细胞
从引擎盖下的冷冻管中取出装有细胞的纸条,并将其放入装有10 ml 完整DMEM的15 ml 离心管中。
手动摇动离心管约20秒钟,以从纸中释放细胞。
用无菌镊子从试管上取下纸带。
将离心管中的细胞悬液在37 °C下以300 x g 离心5分钟以沉淀细胞。
用三倍洗涤细胞沉淀 1 ml 室温DPBS。
将细胞重悬于T75烧瓶中的完整DMEM 中,并将烧瓶置于37 °C 和5%CO 2的加湿培养箱中,以2D培养扩增它们(图2)。
 

图2.共聚焦显微镜图像显示,在基于纸和常规(对照)冷冻保存后,细胞以二维培养物扩增。增殖3天后,肌动蛋白(红色)和微管蛋白(绿色)的扩散以及细胞核(蓝色)的存在证实了无纸化和新鲜培养的HeLa细胞之间没有形态异常或差异。

(可选)使用细胞计数室载玻片和自动细胞计数仪,进行台盼蓝排除试验,直接计算释放的活细胞和死细胞的数量。
(可选)利用其仍然在纸张上的细胞(图3)以进一步在体外三维培养物生长和球体结构(Alnemari 等人,2020)或任何其它细胞应用。这是通过将纸张以及剩余的单元格在无菌培养皿中,让细胞在37 °C和5%CO 2的加湿培养箱内进行3D扩增。请注意,此步骤已在我们最近的研究(Alnemari et al。,2018)中扩展到在纸平台上形成3D细胞培养物的阵列,目前我们正在提高其在冷冻保存这些阵列中的适用性。
 
3D细胞培养物的冷冻保存
在4°C(置于冰上)中将细胞(〜10 7个细胞/ ml 浓度)添加到100%Matrigel中,然后将细胞在Matrigel中的悬浮液吸移到置于无菌陪替氏培养皿中的高压灭菌纸条上(〜10 7个细胞/ m 每厘米2 升)。
在室温下将纸条放置10分钟,以使Matrigel固化。
将纸条在37 °C 和5%CO 2的加湿培养箱中浸入完整的DMEM中7天,以使纸中的细胞增殖。
滚动纸条并将其放入装有DMEM冷冻介质的标准冷冻管中。将冷冻管放入冷冻箱中。
   以1°C min -1的速度将增殖的细胞缓慢冷冻至-80°C过夜。
将细胞转移至-196°C的液氮中以进行长期保存。

图3.共聚焦显微镜图像显示融化后纸张中的活细胞释放。将它们加载到10 µg / ml 纤连蛋白处理过的纸上并随后冷冻和融化后,纸平台内活(绿色)和死(红色)HeLa细胞的相对分布从视觉上证明了活细胞可以从纸上有效释放。

解冻并按需提供3D细胞培养
从液氮中取出冷冻管,并在37 ° C的水浴中融化30 s。
如果将细胞培养物以较大的薄片冷冻保存;切一小片,然后融化。将未使用的液体放回液氮中。
将纸条和剩余细胞浸入无菌陪替氏培养皿中的完整DMEM中,以在37 °C 和5%CO 2的加湿培养箱中进行额外的细胞增殖。
(可选)使用活/死测定法目测评估(作为对照)试纸中活/死细胞的分布(图4)。这是一个有用的步骤,尤其是在处理3D细胞培养物时(有关过程详细信息,请参见食谱)。

数据分析
通过使用绿色(488 nm)和红色(612 nm)激发波长的Olympus FV1000倒置共聚焦激光扫描显微镜(Olympus Corporation)对细胞进行成像,进行活/死分析,并使用Imaris软件进行后处理图像。使用10倍空气物镜在纸上成像最深160 µm。Z堆栈成像以5 µm的增量进行,并使用Imaris软件创建了它们的3D投影。
图4.共聚焦显微镜图像显示融化后基于纸质3D培养的细胞存活。在7天的3D纸质HeLa细胞培养之后,以1°C min -1的速率冷冻过夜至-80 °C ,最后融化;实验表明活细胞的完整性和存活率。

笔记
乳腺癌MCF-7癌细胞系,前列腺PC3癌细胞系和淋巴细胞JKT细胞系(ATCC,目录号分别为CRL-3435,CRL-1435和TIB-152)也可以用于纸质冷冻保存。参见Alnemari 等。(2020)作比较。以下是准备细胞的详细步骤。使用HeLa细胞时,其余的冷冻保存和解冻步骤相同。
对MCF-7细胞使用完整的DMEM,对PC3和JKT细胞使用完整的RPMI培养基,两者均补充有10%(vol / vol)的FBS和1%(vol / vol)的Pen-Strep 。
使用TrypLE将PC3和MCF-7细胞约80%融合后,将其从烧瓶中解离,并以300 x g 离心5分钟。收集悬浮的JKT细胞,并以200 x g 离心7分钟。
将细胞沉淀(〜10 7个细胞)重悬于完全冷冻的培养基中,其中PC3和MCF-7细胞的DMSO浓度为10%(体积/体积),JKT细胞的DMSO浓度为5%(体积/体积)。
室温下纸中水量的变化(水蒸发速率)是每9 cm 2 纸带0.002 g min -1 。
300 µl 细胞悬浮液(浓度为10 7个细胞/ ml )足以使3×3×0.02 cm 3的纸体积充满细胞。

菜谱
完全中等
要准备用于HeLa细胞的完整细胞培养基,请在DMEM中补充10%(vol / vol)FBS和1%(vol / vol)Pen-Strep

冷冻介质
要准备用于HeLa细胞的冷冻培养基,请添加10%(vol / vol)DMSO以完成DMEM

活/死一个SSAY
在5 ml 室温DPBS中稀释2.5 µl 钙黄绿素-AM和10 µl 乙二甲基均二聚体-1
移液管将100μl在工作溶液在纸张上的条纹和温育30分钟的所述暗
将纸张放在盖玻片上,并使用安装介质将其用另一个盖玻片密封
             在显微镜下观察细胞的完整性,存活率和生存力
 
致谢
这项工作得到了纽约大学阿布扎比分校(NYUAD)的财政支持。我们感谢纽约大学阿布扎比核心技术平台的技术支持。该协议是根据Alnemari 等人(2005年)描述的程序进行修订的。(2020)。

利益争夺
作者宣称没有竞争利益。

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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Deliorman, M., Sukumar, P., Alnemari, R. and Qasaimeh, M. A. (2020). A Method to Efficiently Cryopreserve Mammalian Cells on Paper Platforms. Bio-protocol 10(18): e3764. DOI: 10.21769/BioProtoc.3764.
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