BioVector? McCoy 細(xì)胞說明書

BioVector? McCoy Cell Line Manual

第一部分 中文說明

一 產(chǎn)品基本信息與生物學(xué)背景

• 細(xì)胞名稱：McCoy 細(xì)胞（McCoy Cell Line）。

• 菌株編號(hào)與別名：ATCC CRL-1696、ECACC 90010305、DSMZ ACC-625。

• 物種來源：小鼠（Mouse / Mus musculus）。

  • 歷史澄清與科學(xué)修正：McCoy 細(xì)胞系最初在 1957 年被報(bào)道分離自人類患者的滑膜組織。然而，后經(jīng)國際細(xì)胞系認(rèn)證委員會(huì)（ICLAC）以及美國標(biāo)準(zhǔn)生物品收藏中心（ATCC）通過同工酶分析、染色體核型鑒定和 STR（短串聯(lián)重復(fù)序列）基因分型 嚴(yán)格證實(shí)，原始的人源 McCoy 細(xì)胞早在早期傳代中已被小鼠 L-929 細(xì)胞（成纖維細(xì)胞）完全污染并替代。因此，目前全球各大細(xì)胞庫（ATCC, ECACC）現(xiàn)存分發(fā)的 McCoy 細(xì)胞，其本質(zhì)均為小鼠源性細(xì)胞。

• 細(xì)胞類型與形態(tài)：成纖維細(xì)胞樣（Fibroblast-like），貼壁生長（Adherent）。

• 生物安全級(jí)別：1級(jí)（BSL-1）。細(xì)胞已通過嚴(yán)格的常見病原體與支原體篩查，呈嚴(yán)格陰性。

二 核心科研價(jià)值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

雖然其遺傳學(xué)背景為小鼠成纖維細(xì)胞，但 McCoy 細(xì)胞在醫(yī)學(xué)微生物學(xué)和病毒學(xué)領(lǐng)域具有極其特殊且不可替代的“宿主細(xì)胞”地位：

1. 衣原體（Chlamydia）體外診斷與分離培養(yǎng)（標(biāo)桿模型）：McCoy 細(xì)胞是全球臨床和科研公認(rèn)用于分離、培養(yǎng)和鑒定沙眼衣原體（Chlamydia trachomatis）和肺炎衣原體（Chlamydia pneumoniae）的標(biāo)準(zhǔn)宿主細(xì)胞。利用經(jīng)環(huán)己酰亞胺（Cycloheximide）或射線照射處理的 McCoy 細(xì)胞，能夠顯著抑制宿主細(xì)胞自身的 DNA 合成，從而極大地促進(jìn)衣原體原體（EB）向網(wǎng)織體（RB）的轉(zhuǎn)化及胞內(nèi)包涵體（Inclusions）的形成。

2. 專性胞內(nèi)寄生菌與微需氧菌協(xié)同培養(yǎng)：如前所述，McCoy 細(xì)胞也是培養(yǎng)諸如胞內(nèi)勞森菌（Lawsonia intracellularis）等獸醫(yī)及比較醫(yī)學(xué)領(lǐng)域中專性胞內(nèi)寄生菌的關(guān)鍵體外活體細(xì)胞基質(zhì)。

3. 毒素生物學(xué)活性檢測：廣泛用于檢測和定量分析艱難梭菌（Clostridioides difficile）產(chǎn)生的毒素 A（TcdA）和毒素 B（TcdB）的細(xì)胞毒性（Cytotoxicity Assay），觀察其引起的細(xì)胞變圓（Rounding）等病理形態(tài)改變。

三 實(shí)驗(yàn)室細(xì)胞復(fù)蘇、擴(kuò)增傳代與冷凍保存標(biāo)準(zhǔn)步驟

1. 完全培養(yǎng)基配置（Complete Growth Medium）

McCoy 細(xì)胞對(duì)營養(yǎng)條件要求相對(duì)常規(guī)，但為了保證其作為宿主細(xì)胞時(shí)的強(qiáng)健狀態(tài)，建議配置如下體系：

• 基礎(chǔ)培養(yǎng)基：EMEM（推薦） 或 高糖 DMEM。

• 完全添加劑成分：

  • 10% 優(yōu)質(zhì)胎牛血清（FBS）。

  • 1% 滅菌雙抗（Penicillin-Streptomycin）。

  • 注：若后續(xù)實(shí)驗(yàn)用于衣原體培養(yǎng)，接種衣原體后的維持液通常需要去除雙抗或更換為專用抗生素配方，以防抑制衣原體生長。

2. 細(xì)胞復(fù)蘇（Thawing Protocol）

1. 將配置好的完全培養(yǎng)基在 37 攝氏度水浴中預(yù)熱。

2. 從液氮罐中取出 McCoy 細(xì)胞凍存管，立即投入 37 攝氏度恒溫水浴箱中，輕微晃動(dòng)。

3. 在 1 分鐘內(nèi)令其急速融化。迅速用 75% 酒精擦拭外部消毒。

4. 在生物安全柜內(nèi)，將細(xì)胞懸液吸出，置于含有 5 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，極其輕柔地顛倒混勻。

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

6. 加入 5 mL 新鮮完全培養(yǎng)基重懸細(xì)胞，接種于培養(yǎng)瓶中，置于 37 攝氏度、5% $CO_2$ 孵箱中培養(yǎng)。次日觀察貼壁情況。

3. 細(xì)胞傳代（Passaging / Subculture）

• 傳代時(shí)機(jī)：McCoy 細(xì)胞生長較快，當(dāng)細(xì)胞密度達(dá)到約 80% - 90% 融合度時(shí)必須執(zhí)行傳代。避免細(xì)胞 100% 堆疊生長，否則會(huì)導(dǎo)致細(xì)胞狀態(tài)下滑。

• 傳代步驟：

  1. 吸除舊培養(yǎng)基，用無菌 PBS（不含鈣鎂離子）洗滌細(xì)胞表面 1-2 次。

  2. 加入適量 0.25% Trypsin-EDTA 消化液，確保覆蓋細(xì)胞層。

  3. 置于 37 攝氏度孵箱中消化 1 至 2 分鐘。在顯微鏡下觀察，當(dāng)成纖維樣細(xì)胞變圓、細(xì)胞間隙增大并開始脫離瓶壁時(shí)，立即加入 2 倍體積的含血清完全培養(yǎng)基終止消化。

  4. 輕柔吹打瓶壁使細(xì)胞完全脫落。收集至離心管中，300 × g 離心 3 分鐘，棄上清。

  5. 按照 1:3 至 1:6 的傳代比例接種到新的培養(yǎng)瓶中。通常 2-3 天即可再次長滿。

4. 細(xì)胞冷凍保存（Cryopreservation）

• 凍存液配方：90% 完全培養(yǎng)基（或純 FBS） + 10% DMSO。

• 凍存操作：收集處于對(duì)數(shù)生長活躍期的健康細(xì)胞，離心棄上清。調(diào)整細(xì)胞密度至 $1 \times 10^6$ 至 $5 \times 10^6$ cells/vial。加入凍存液重懸分裝后，立即投入標(biāo)準(zhǔn)程序降溫盒（梯度降溫盒，1 攝氏度/min），置于 -80 攝氏度過夜，次日必須轉(zhuǎn)移至 -196 攝氏度液氮中長期冷凍保存。

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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