BioVector® 人牙囊干细胞说明书

BioVector® Human Dental Follicle Stem Cells (hDFSCs) 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：人牙囊干细胞（Human Dental Follicle Stem Cells, hDFSCs）。

• 物种来源：人源（Human），通常分离自正畸需要拔除的年轻患者（如 12-18 岁）健康阻生智齿（第三磨牙）外周包裹的牙囊组织。

• 核心生物学特性：

  • 外胚层间充质干细胞（ECT-MSCs）：牙囊是包绕在发育中牙胚外周的疏松结缔组织囊，来源于颅神经嵴外胚间充质。hDFSCs 属于外胚层来源的间充质干细胞，不仅具备传统间充质干细胞（MSCs）的自我更新和多向分化潜能，还保留了神经外胚层的部分分化特性。

  • 免疫表型特征：根据国际细胞治疗协会（ISCT）标准，hDFSCs 稳定高表达间充质干细胞表面标志物（如 CD73, CD90, CD105, CD29, CD44），同时高度表达胚胎干细胞及外胚层标志物（如 Stro-1, Notch-1, Nestin）；而造血干细胞及内皮标志物（如 CD34, CD45, CD11b, HLA-DR）表达呈严格阴性。

  • 多向分化潜能：在特定的体外诱导条件下，hDFSCs 能够高效向成牙骨质细胞（Cementoblasts）、成骨细胞（Osteoblasts）、成牙本质细胞（Odontoblasts）分化，且具备跨生殖层向神经样细胞（Neurogenic cells）和脂肪细胞分化的能力。

• 生长特性：贴壁生长（Adherent），主要呈现长梭形、成纤维细胞样（Fibroblast-like）的集落状（CFU-F）生长形态。

• 生物安全级别：1级（BSL-1）。细胞已通过严格的支原体、原虫、细菌、真菌及人类核心病毒（HIV, HBV, HCV）筛查，结果均为阴性。

二 核心科研价值与转化医学应用

人牙囊干细胞被公认为牙齿再生医学及组织工程领域中最具潜力的“种子细胞”之一：

1. 牙周组织再生与功能性生物牙根构建：牙囊在体内原本就是牙周组织（牙骨质、牙周膜和牙槽骨）的发育前体。hDFSCs 是目前体外研究牙骨质再生、牙周膜（PDL）重塑以及构建功能性成形生物牙根（Biomimetic Root）的核心细胞模型。

2. 颅面部骨缺损修复修复（Bone Tissue Engineering）：hDFSCs 的成骨分化能力极强，常与各类生物相容性支架材料（如胶原、羟基磷灰石 HA、聚乳酸等）结合，用于研究体外颌骨缺森重建以及干细胞-材料交互动力学。

3. 神经再生与神经退行性疾病研究：由于其特有的神经嵴来源背景，hDFSCs 在受到神经诱导时能快速上调 Nestin、Tuj1 和 GFAP 的表达。常用于体外研究轴突导向、神经保护作用以及周围神经损伤修复的细胞治疗模型。

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）

hDFSCs 属于对生长因子需求较高的原代及有限传代干细胞，建议配置如下高营养体系：

• 基础培养基：alpha-MEM（推荐） 或 DMEM/F12（含低糖）。

• 完全添加剂成分：

  • 10% - 15% 优质优质胎牛血清（FBS，建议选择干细胞专属级别）。

  • 1% 灭菌双抗（Penicillin-Streptomycin）。

  • 任选添加剂：部分原代早期培养为了加速扩增，可添加 2-4 mM L-谷氨酰胺及 1% 非必需氨基酸（NEAA），但常规培养上述配方已足够。

2. 细胞复苏（Thawing Protocol）

1. 将完全培养基放入 37摄氏度 水浴中预热。

2. 从液氮罐中取出 hDFSCs 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（至仅剩极小冰芯时捞出）。迅速用酒精擦拭外部。

4. 在生物安全柜内，用移液管将细胞悬液吸出，置于含有 5-8 mL 预热完全培养基的 15 mL 离心管中，极其轻柔地颠倒混匀，以稀释 DMSO 浓度。

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

6. 加入 3-5 mL 新鲜完全培养基，用移液枪极轻柔地吹打 1-2 次使细胞重悬（严禁剧烈震荡导致刚复苏的干细胞机械损伤）。接种于培养瓶中，置于 37摄氏度、5% CO2 孵箱中培养。

3. 细胞传代（Passaging / Subculture）

• 传代时机：切勿让干细胞长至 100% 完全融合。当细胞密度达到约 80% - 85% 融合度时必须执行传代。干细胞过密会导致接触抑制，引发自发性分化或丧失干性。

• 传代步骤：

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

  2. 加入适量 0.25% Trypsin-EDTA 消化液（一般 T25 瓶加 1-1.5 mL），确保覆盖细胞。

  3. 置于 37摄氏度 孵箱中消化 1 至 3 分钟。在显微镜下观察，当大块的长梭形细胞变圆并开始收缩、部分脱离瓶壁时，立即加入 2 倍体积的含血清完全培养基终止消化。

  4. 用移液枪轻柔吹打瓶壁，使贴壁细胞完全脱落。将细胞悬液收集至离心管中，300 x g 离心 3 分钟，弃上清。

  5. 按照 1:2 至 1:3 的传代比例接种到新的培养瓶/皿中。注：原代间充质干细胞传代比例不宜过稀，以保持细胞间的信号通讯。

4. 细胞冷冻保存（Cryopreservation）

• 冻存液配方：55% 基础培养基 + 40% 优质胎牛血清（FBS） + 5% DMSO；或采用 90% FBS + 10% DMSO。推荐使用市售无血清的高效专用干细胞冻存液以维持极高的复苏活率。

• 冻存操作：收集处于对数生长活跃期（传代代数通常控制在 P3-P6 之间最佳）的健康细胞，离心弃上清。调整细胞密度至 $\ge 1 \times 10^6$ cells/vial。加入冻存液重悬分装后，立即投入标准程序降温盒（异丙醇梯度降温盒，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: Human Dental Follicle Stem Cells (hDFSCs).

• Species Origin: Human. Isolated from healthy loose connective tissue of the dental follicle encapsulating impacted third molars (wisdom teeth), typically harvested during routine orthodontic extractions from adolescent patients (aged 12–18 years).

• Core Biological Framework:

  • Ectomesenchymal Stem Cell Lineage: The dental follicle is a specialized ectomesenchymal tissue derived from the cranial neural crest during odontogenesis. Belonging to the ectomesenchymal category, hDFSCs retain conventional mesenchymal stem cell (MSC) properties coupled with neural crest-derived developmental plasticity.

  • Immunophenotypic Profile: In alignment with the International Society for Cellular Therapy (ISCT) consensus, hDFSCs consistently exhibit high expressions of MSC surface markers (CD73, CD90, CD105, CD29, CD44) and prominent neural crest/embryonic stem signs (Stro-1, Notch-1, Nestin). Conversely, hematopoietic and endothelial markers (such as CD34, CD45, CD11b, and HLA-DR) are strictly negative.

  • Multi-lineage Differentiation Plasticity: Under tailored biochemical induction parameters in vitro, hDFSCs can easily commit to lineage-specific pathways yielding cementoblasts, osteoblasts, and odontoblasts, alongside cross-germline potential into neurogenic cells and adipocytes.

• Growth Topology: Adherent growth mode. Displays typical spindle-shaped, elongated, fibroblast-like morphologies exhibiting prominent Colony-Forming Unit-Fibroblast (CFU-F) clustering traits.

• Biosafety Matrix: Biosafety Level 1 (BSL-1). Rigorously authenticated and pre-screened negative for mycoplasma, protozoa, bacterial/fungal pathobionts, and core human viral indicators (HIV, HBV, HCV).

II Strategic Research Value & Translational Fields

Human dental follicle stem cells are widely recognized as choice seed cell matrices in structural tooth organ engineering and general regenerative medicine:

1. Periodontal Regeneration and Bio-Root Fabrication: In vivo, the dental follicle naturally yields the definitive components of the periodontium (cementum, periodontal ligament [PDL], and alveolar bone). In vitro, hDFSCs serve as a premier platform to examine cementogenesis, physiological PDL shearing integration, and functional biomimetic dental root tissue morphogenesis.

2. Craniofacial Bone Tissue Engineering: Benefiting from their vigorous osteogenic capabilities, hDFSCs are regularly integrated with complex biocompatible matrices (such as collagen, hydroxyapatite [HA], and PLA scaffolds) to model maxillo-mandibular bone reconstruction and dissect stem cell-material interfacial kinetics.

3. Neurogenesis & Neurodegenerative Therapy: Owing to their neural crest ontogeny, hDFSCs rapidly upgrade Nestin, Tuj1, and GFAP expression patterns under neurogenic induction. They provide a viable human-derived somatic model to explore axonal guidance, local neuroprotection, and peripheral nerve injury cell-based therapeutics.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Complete Growth Medium

As primary and low-passage progenitor lines sensitive to growth factor exhaustion, hDFSCs thrive optimally within highly enriched nutritional parameters:

• Basal Medium: alpha-MEM (Highly Recommended) or low-glucose DMEM/F12 alternative.

• Complete Media Supplements:

  • 10% to 15% Premium Fetal Bovine Serum (FBS, Stem-Cell Qualified Grade Preferred).

  • 1% Penicillin-Streptomycin cocktail.

  • Optional Additives: Early primary expansions may include 2–4 mM L-glutamine and 1% Non-Essential Amino Acids (NEAA) to accelerate log-phase velocity; however, standard cultivation runs efficiently within the core formula noted above.

2. Cryovial Thawing Routine

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

2. Retrieve the hDFSCs cryovial from liquid nitrogen storage and instantly submerge it into the 37 degree Celsius water bath with continuous gentle agitation.

3. Complete the thawing cycle rapidly within 1 minute (extract when a minute ice core remains). Swab the exterior thoroughly with 70% ethanol.

4. In a biosafety cabinet, transfer the cell slurry into a sterile 15 mL conical tube pre-filled with 5–8 mL of pre-warmed complete growth medium. Mix by gentle inversion to cushion osmotic pressure variations caused by liquefied DMSO.

5. Centrifuge the suspension at 200–300 x g for 3–5 minutes, then aspirate the DMSO-contaminated supernatant.

6. Replenish with 3–5 mL of fresh complete medium, and gently pipette up and down 1–2 times to re-suspend the pellet. Do not vortex or aggressively pipette to avoid mechanical shearing of vulnerable thawed cells. Plate into culture vessels and incubate at 37 degree Celsius under a humidified 5% CO2 atmosphere.

3. Subculturing and Cell Passaging Guide

• Confluency Threshold: Do not allow the cultures to reach 100% complete confluency. Execute passaging promptly when the monolayer strikes 80%–85% confluency. Over-crowding triggers contact inhibition, causing spontaneous lineage differentiation or irreversible loss of stemness features.

• Step-by-Step Harvesting:

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

  2. Dispense an adequate volume of 0.25% Trypsin-EDTA Solution across the cell matrix (approx. 1–1.5 mL for standard T25 flasks).

  3. Incubate at 37 degree Celsius for 1 to 3 minutes. Monitor under an inverted microscope; as soon as the elongated spindle cells round up, shrink, and begin separating from the baseline matrix, immediately add a double volume of serum-containing complete growth medium to quench the enzyme.

  4. Resuspend the layer by gentle pipetting to dislodge remaining cells. Transfer the slurry to a conical tube and centrifuge at 300 x g for 3 minutes, then discard the supernatant.

  5. Re-plate into fresh flasks at a standard split ratio of 1:2 to 1:3. Note: Avoid plating primary MSCs at over-diluted ratios to preserve essential local paracrine signaling loops.

4. Cryopreservation Protocol

• Freezing Medium Matrix: 55% Basal Medium + 40% FBS + 5% DMSO, or 90% FBS + 10% DMSO. Alternatively, deploy validated protein-free commercial cryopreservation configurations for stem cells to maximize post-thaw recovery parameters.

• Freezing Routine: Harvest healthy cells strictly during their log-growth window (optimally utilizing passages P3 to P6). Spin down, discard the supernatant, and adjust the cell density to $\ge 1 \times 10^6$ viable cells/vial. Aliquot into cryovials and instantly place inside a standardized isopropyl alcohol controlled-rate freezing container. Store at -80 degree Celsius overnight, and relocate the tubes into liquid nitrogen (-196 degree Celsius) the following day for indefinite preservation.

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

电话:400-800-2947

工作QQ/微信同号:1843439339

网址http://www.biovector.net