BioVector® McCoy 细胞说明书

BioVector® McCoy Cell Line Manual

第一部分 中文说明

一 产品基本信息与生物学背景

• 细胞名称：McCoy 细胞（McCoy Cell Line）。

• 菌株编号与别名：ATCC CRL-1696、ECACC 90010305、DSMZ ACC-625。

• 物种来源：小鼠（Mouse / Mus musculus）。

  • 历史澄清与科学修正：McCoy 细胞系最初在 1957 年被报道分离自人类患者的滑膜组织。然而，后经国际细胞系认证委员会（ICLAC）以及美国标准生物品收藏中心（ATCC）通过同工酶分析、染色体核型鉴定和 STR（短串联重复序列）基因分型 严格证实，原始的人源 McCoy 细胞早在早期传代中已被小鼠 L-929 细胞（成纤维细胞）完全污染并替代。因此，目前全球各大细胞库（ATCC, ECACC）现存分发的 McCoy 细胞，其本质均为小鼠源性细胞。

• 细胞类型与形态：成纤维细胞样（Fibroblast-like），贴壁生长（Adherent）。

• 生物安全级别：1级（BSL-1）。细胞已通过严格的常见病原体与支原体筛查，呈严格阴性。

二 核心科研价值与转化医学应用

虽然其遗传学背景为小鼠成纤维细胞，但 McCoy 细胞在医学微生物学和病毒学领域具有极其特殊且不可替代的“宿主细胞”地位：

1. 衣原体（Chlamydia）体外诊断与分离培养（标杆模型）：McCoy 细胞是全球临床和科研公认用于分离、培养和鉴定沙眼衣原体（Chlamydia trachomatis）和肺炎衣原体（Chlamydia pneumoniae）的标准宿主细胞。利用经环己酰亚胺（Cycloheximide）或射线照射处理的 McCoy 细胞，能够显著抑制宿主细胞自身的 DNA 合成，从而极大地促进衣原体原体（EB）向网织体（RB）的转化及胞内包涵体（Inclusions）的形成。

2. 专性胞内寄生菌与微需氧菌协同培养：如前所述，McCoy 细胞也是培养诸如胞内劳森菌（Lawsonia intracellularis）等兽医及比较医学领域中专性胞内寄生菌的关键体外活体细胞基质。

3. 毒素生物学活性检测：广泛用于检测和定量分析艰难梭菌（Clostridioides difficile）产生的毒素 A（TcdA）和毒素 B（TcdB）的细胞毒性（Cytotoxicity Assay），观察其引起的细胞变圆（Rounding）等病理形态改变。

三 实验室细胞复苏、扩增传代与冷冻保存标准步骤

1. 完全培养基配置（Complete Growth Medium）

McCoy 细胞对营养条件要求相对常规，但为了保证其作为宿主细胞时的强健状态，建议配置如下体系：

• 基础培养基：EMEM（推荐） 或 高糖 DMEM。

• 完全添加剂成分：

  • 10% 优质胎牛血清（FBS）。

  • 1% 灭菌双抗（Penicillin-Streptomycin）。

  • 注：若后续实验用于衣原体培养，接种衣原体后的维持液通常需要去除双抗或更换为专用抗生素配方，以防抑制衣原体生长。

2. 细胞复苏（Thawing Protocol）

1. 将配置好的完全培养基在 37 摄氏度水浴中预热。

2. 从液氮罐中取出 McCoy 细胞冻存管，立即投入 37 摄氏度恒温水浴箱中，轻微晃动。

3. 在 1 分钟内令其急速融化。迅速用 75% 酒精擦拭外部消毒。

4. 在生物安全柜内，将细胞悬液吸出，置于含有 5 mL 预热完全培养基的 15 mL 离心管中，极其轻柔地颠倒混匀。

5. 以 200-300 × g 离心 3 分钟，小心吸除含有 DMSO 的上清液。

6. 加入 5 mL 新鲜完全培养基重悬细胞，接种于培养瓶中，置于 37 摄氏度、5% $CO_2$ 孵箱中培养。次日观察贴壁情况。

3. 细胞传代（Passaging / Subculture）

• 传代时机：McCoy 细胞生长较快，当细胞密度达到约 80% - 90% 融合度时必须执行传代。避免细胞 100% 堆叠生长，否则会导致细胞状态下滑。

• 传代步骤：

  1. 吸除旧培养基，用无菌 PBS（不含钙镁离子）洗涤细胞表面 1-2 次。

  2. 加入适量 0.25% Trypsin-EDTA 消化液，确保覆盖细胞层。

  3. 置于 37 摄氏度孵箱中消化 1 至 2 分钟。在显微镜下观察，当成纤维样细胞变圆、细胞间隙增大并开始脱离瓶壁时，立即加入 2 倍体积的含血清完全培养基终止消化。

  4. 轻柔吹打瓶壁使细胞完全脱落。收集至离心管中，300 × g 离心 3 分钟，弃上清。

  5. 按照 1:3 至 1:6 的传代比例接种到新的培养瓶中。通常 2-3 天即可再次长满。

4. 细胞冷冻保存（Cryopreservation）

• 冻存液配方：90% 完全培养基（或纯 FBS） + 10% DMSO。

• 冻存操作：收集处于对数生长活跃期的健康细胞，离心弃上清。调整细胞密度至 $1 \times 10^6$ 至 $5 \times 10^6$ cells/vial。加入冻存液重悬分装后，立即投入标准程序降温盒（梯度降温盒，1 摄氏度/min），置于 -80 摄氏度过夜，次日必须转移至 -196 摄氏度液氮中长期冷冻保存。

Part 2 English Section

I General Information and Biological Background

• Cell Line Name: McCoy Cell Line.

• Strain Designations and Aliases: ATCC CRL-1696, ECACC 90010305, DSMZ ACC-625.

• Species Origin: Mouse (Mus musculus).

  • Historical Clarification & Scientific Correction: The McCoy cell line was originally reported in 1957 as being isolated from human synovial tissue fragments. However, subsequent intensive investigation by the International Cell Line Authentication Committee (ICLAC) and the ATCC utilizing isoenzyme analysis, karyotyping, and STR (Short Tandem Repeat) profiling definitively demonstrated that the original human cells were entirely cross-contaminated and replaced by mouse L-929 fibroblasts during early serial passaging. Consequently, all certified stocks maintained by global repositories are bona fide mouse lineages.

• Cell Type and Topography: Fibroblast-like; exhibits strictly adherent growth properties.

• Biosafety Matrix: Biosafety Level 1 (BSL-1). Validated and pre-screened negative for mycoplasma, standard bacterial/fungal pathobionts, and common murine viral agents.

II Strategic Research Value and Translational Fields

Despite its structural identity as a mouse fibroblast, the McCoy cell line occupies an essential paradigm in diagnostic microbiology and cellular virology as an indispensable "host matrix":

1. Gold Standard Host for Chlamydia Cultivation: McCoy cells are globally cross-referenced as the foundational substrate for the in vitro isolation, propagation, and diagnostic tracking of Chlamydia trachomatis and Chlamydia pneumoniae. Pre-treatment of McCoy monolayers with cycloheximide or gamma irradiation stalls host cell nuclear division without inducing toxicity, diverting the metabolic machinery to optimize Chlamydia elementary body (EB) into reticulate body (RB) transition, thereby amplifying intracellular inclusion body yields.

2. Co-culture Substrate for Obligate Intracellular Pathogens: Serves as a vital cellular monolayer for sustaining fastidious obligate intracellular veterinary bacteria, such as Lawsonia intracellularis, which fail to divide outside a functional eukaryotic cytoplasmic niche.

3. Clostridial Cytotoxicity Bioassays: Extensively integrated into clinical diagnostic pipelines to detect and quantify the functional kinetics of Clostridioides difficile Toxin A (TcdA) and Toxin B (TcdB) via definitive cell-rounding morphologic readout assays.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Complete Growth Medium

McCoy cells are robust and maintain highly predictable doubling kinetics under standard formulation profiles:

• Basal Medium: Minimum Essential Medium (EMEM - Highly Recommended) or high-glucose DMEM alternative.

• Complete Media Supplements:

  • 10% Premium Fetal Bovine Serum (FBS).

  • 1% Penicillin-Streptomycin cocktail.

  • Technical Note: If monolayers are dedicated to subsequent Chlamydia infection setups, the post-inoculation maintenance medium must exclude routine antibiotics to prevent unintended inhibition of bacterial development.

2. Cryovial Thawing Routine

1. Pre-warm the formulated complete growth medium within a 37 degree Celsius water bath.

2. Extract the McCoy cryovial from liquid nitrogen and instantly plunge it into the 37 degree Celsius water bath with continuous gentle agitation.

3. Achieve complete liquefaction rapidly within 1 minute. Swab the exterior thoroughly with 70% ethanol.

4. Inside a biosafety enclosure, pipette the cell suspension directly into a 15 mL conical tube filled with 5 mL of pre-warmed complete growth medium. Mix by gentle inversion.

5. Centrifuge the suspension at 200–300 × g for 3 minutes, then cleanly aspirate the DMSO-laden supernatant.

6. Replenish with 5 mL of fresh complete medium, gently resuspend the pellet, transfer into the target culture flask, and incubate at 37 degree Celsius under a humidified 5% $CO_2$ atmosphere.

3. Subculturing and Cell Passaging Guide

• Confluency Threshold: McCoy cells exhibit highly active division kinetics. Subculturing must be performed precisely when the monolayer hits 80%–90% confluency. Do not allow cells to remain at 100% saturation to avoid contact inhibition and structural deterioration.

• Step-by-Step Harvesting:

  1. Aspirate the spent culture medium and wash the layer 1–2 times with sterile, calcium/magnesium-free PBS.

  2. Add an appropriate volume of 0.25% Trypsin-EDTA Solution to completely submerge the cell sheet.

  3. Incubate at 37 degree Celsius for 1 to 2 minutes. Monitor under an inverted microscope; as soon as the fibroblast-like cells round up, retract, and release from the substrate, immediately add a double volume of complete serum-supplemented medium to halt enzymatic degradation.

  4. Gently pipette the vessel walls to yield a single-cell suspension. Spin down at 300 × g for 3 minutes and discard the supernatant.

  5. Re-plate into fresh culture vessels utilizing a standard split ratio ranging from 1:3 to 1:6. Monolayers typically reach optimal saturation within 48–72 hours.

4. Cryopreservation Protocol

• Freezing Medium Matrix: 90% Complete Growth Medium (or pure premium FBS) supplemented with 10% analytical-grade DMSO.

• Freezing Routine: Harvest healthy, log-phase cells showing optimal viability profiles. Centrifuge, discard the supernatant fluid, and re-suspend the cell mass to log a final target density of $1 \times 10^6$ to $5 \times 10^6$ viable cells/vial. Aliquot into sterile cryovials and transfer immediately to a standardized isopropyl alcohol controlled-rate freezing box. Store at -80 degree Celsius overnight, and shift the vials into liquid nitrogen (-196 degree Celsius) the following day for indefinite preservation.

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