SF27 (不含维生素A, 50×)，是一种优化的无血清添加剂，广泛用于胚胎，出生后和成体的海马神经元和其他中枢神经系统（CNS）神经元的生长、分化和活性维持。 无论是悬浮的神经球还是贴壁单层培养，都是用于培养神经祖细胞和神经干细胞的理想选择，而不会诱导分化。此款 SF27 添加剂为不含维生素 A 的 SF27 完全添加剂。这种去除了维生素 A 的添加剂可防止干细胞向神经细胞分化。 SF27 添加剂（不含维生素 A）以50×液体形式提供在神经元基础培养基中使用 SF27 添加剂，用于培养产前和胚胎神经元，以获得优化的活性和长期的存活率。也可用于培养神经来源的肿瘤细胞系，可有效地保证其活性。现也已广泛应用于各种乳腺癌、小肠、胃、肝脏等类器官的培养中。
应用：
用于大鼠海马神经元及多种中枢神经元细胞的生长和活性维持；干细胞增殖及分化；维持神经细胞来源的肿瘤细胞系的活性；优化胚胎和出生后神经元的活性和长期存活率；胚胎干细胞分化为神经元谱系(神经元和星形胶质细胞)；肿瘤类器官的培养。
使用方法：
SF27 (不含维生素A, 50×) 是 50×浓度。使用前请用基础培养基稀释到 1× 。如准备 50 mL 培养基： 将 1 mL 的 SF27 无血清添加剂 ( 不含维生素A ，50×) 加入到 49 mL 的基础培养基中。
培养基的配置：
由于 L-谷氨酰胺在水溶液中不稳定，其分解产物对细胞具有毒性，神经元基础培养基和 B-27中都未包含 L-谷氨酰胺，因此在配制培养基时须另外添加 L- 谷氨酰胺。
1、置于4 ℃解冻本产品。
2、在使用前无菌添加 2％本产品和0.5 ML-谷氨酰胺至神经元基础培养基，配制成神经元完全培养基（注：下文中出现的“神经元完全培养基”即指此种添加B-27添加剂和L-谷氨酰胺的神经元基础培养基）。
3、注意：剩余的本产品可以等分成工作体积，并储存在-20 ℃。在以后的试验中根据需要解冻相应体积的本产品。避免反复冻融本产品。
4、对于原代海马神经元培养，神经元完全培养基（由前述步骤制备）需要额外补充25 μM的 L-谷氨酸，在培养的第4天以后的换液中应不再添加谷氨酸。配置好的完全培养基，可于 2-8 ℃的避光保存一周。
细胞培养步骤：
下列步骤已在大鼠胎儿原代皮层神经元中测试。
1、用无菌的冷的 0.05 mg/L 多聚赖氨酸水溶液包被培养表面（玻璃或细胞培养级塑料），每平方厘米表面使用 0.15 mL，37 ℃ 放置过夜 (16 h)；
2、去除多聚赖氨酸溶液，并用无菌蒸馏水冲洗两次（须彻底清洗，因为多聚赖氨酸对细胞有毒性）。在超净工作台中打开培养板的盖子通风，直到每个孔都完全干燥。培养板干燥后可以立即使用或在 4 ℃ 最多保存 2 周。
3、根据实验室标准操作流程从胚胎 16-18 days 的大鼠中分离获得原代海马/皮层神经元细胞，或参考细胞株提供的说明书解冻冻存的原代大鼠神经元细胞。
4、将神经元完全培养基在 37 °C 的水浴锅中预热，建议细胞接种密度为 2×10⁵ 个/孔（24 孔板为例）或根据自身实验对密度进行调整。
5、在将接种了细胞的培养皿放置于 37ºC，5% CO₂ 的培养箱中培养。
6、在接种 16-24 h 后，更换一半体积的新鲜的完全培养基，之后 2-3 days 换液。
分离原代大鼠胚胎神经元：
下列程序推荐用于培养受精 18 天的大鼠胚胎海马神经元和大脑皮层神经元。
1、从受精 18 天的大鼠胚胎中分离大脑皮层和双侧海马。
2、在预置了完全培养基的锥形管中收集所有的组织。放置，直到所有的组织都已解体。
3、使组织沉积在管的底部，然后小心地去除上清液，只留下刚好可覆盖组织的最少量的培养基。
4、在不含钙离子的培养基中，使用 2 mg/L 过滤灭菌的木瓜蛋白酶溶液，在 37 °C 下酶解组织约 30 min，期间轻摇锥形管以帮助降解，5 min/次。每对海马组织使用 2 mL 酶溶液。
5、加入两倍体积的完全培养基以恢复二价阳离子的浓度，停止酶解。
6、使未解离的组织沉降至管底（约 2 min），然后把上清液转移到 15 mL 离心管中，离心，150×g，5 min。
7、在 1 mL 神经元完全培养基中重悬沉淀物，取 10 μL 进行细胞计数。后续的操作按照“复苏和培养冻存的神经元” 的第 8-10 步骤进行。 ·
复苏和培养冻存的神经元：
1、在解冻细胞之前，用神经元完全培养基冲洗无菌的 15 mL 锥形管，然后在超净工作台中通风晾干。
2、从液氮中取出冻存管时可稍微拧松管帽以释放压力，然后再拧紧。
3、在 37 °C 水浴中轻柔搅拌冻存管以快速解冻细胞（小于 2 min）。当观察到冻存管中只有很少量的冰晶存在时（触摸冻存管仍然感觉是冷的）从水浴中取出。
4、在工作台台面上轻敲冻存管以使管内液体沉降到管底。使用事先用神经元完全培养基冲洗并干燥过的 1 mL 吸头极其轻柔地将细胞转移到事先冲洗并干燥的 15 mL 锥形管中。
5、用 1 mL 预热的神经元完全培养基冲洗冲洗冻存管，以每秒一滴的速度极其缓慢地加入到 15 mL 锥形管中的细胞里。每加一滴都轻柔地转动锥形管一次。不要一次即把全部培养基加到锥形管中。
6、慢慢地逐滴加入另外 2 mL 预热的完全培养基，使管内的液体体积为 4 mL。用 1 mL 吸头轻柔地混合液体，注意不要造成任何气泡。
7、用一个事先冲洗干燥过的吸头吸取 10 μL 细胞悬液加入到含有 10 μL ，0.4 %台盼蓝的微量离心管中，轻敲管壁以混匀溶液。使用手动的细胞计数方法测定活细胞密度。解冻的细胞的活率应超过 50 %。
8、在已经事先用多聚赖氨酸包被（参见“细胞培养步骤”）的 24 孔板中每孔中接种大约 2×10⁵ 个细胞或按所需的细胞密度接种。加入预热的神经元完全培养基将细胞悬液稀释到每孔 500 μL。按照“细胞培养步骤”中的 5-6 步骤进行神经元的培养。在 37 °C，含5 % CO₂ 的培养箱中培养。
重要提示：
原代神经元细胞将会贴附在裸露的塑料或玻璃表面，建议事先用神经元完全培养基冲洗所有的塑料和玻璃器 皿的内表面，以获得最高的回收率和产量。由于细胞从冻存状态复苏时极其脆弱，请在整个操作过程中不要震荡或离心细胞。 我们建议每次只解冻一管细胞。务必减少从液氮中转移冻存管到 37 °C 水浴中的操作时间。
注意事项：
1、本产品在使用前在4 ℃过夜解冻。
2、使用样品时将产品充分混匀进行添加。
3、使用样品时应注意无菌操作，避免污染。
4、为保持产品的最佳使用效果，不宜将其过夜放置于室温或较高的温度环境中。
5、请于保质期内使用本产品，超出保质期，请放弃使用。
SF27 (without Vitamin A, 50×) is an optimized serum-free supplement. It is widely applied to support the growth, differentiation and viability maintenance of embryonic, postnatal and adult hippocampal neurons, as well as other central nervous system (CNS) neurons. It is an ideal choice for the culture of neural progenitor cells and neural stem cells in both suspended neurosphere culture and adherent monolayer culture, without inducing cell differentiation. This SF27 supplement is a complete formulation without vitamin A. Vitamin A-depleted supplement can prevent spontaneous differentiation of stem cells into neural lineages. SF27 Supplement ( (without Vitamin A ) is supplied as a 50× liquid formulation. When supplemented into neuronal basal medium, SF27 can be used for the culture of prenatal and embryonic neurons to achieve optimized cell viability and long-term survival. It can also be used for the culture of neural-derived tumor cell lines to effectively maintain cell viability. Currently, it has been widely used in the culture of various organoids, including breast cancer, small intestine, stomach and liver organoids.
Application
Used for the growth and viability maintenance of rat hippocampal neurons and various central nervous system neurons; for the proliferation and differentiation of stem cells; for maintaining the viability of tumor cell lines derived from neural cells; for optimizing the viability and long-term survival rate of embryonic and postnatal neurons; for the differentiation of embryonic stem cells into neuronal lineages (neurons and astrocytes); and for the culture of tumor organoids.
Method of Use
SF27 (without Vitamin A, 50×) is a 50× concentrated solution. Dilute it to 1× with basal medium before use. To prepare 50 mL of medium: add 1 mL of SF27 serum-free supplement (without Vitamin A, 50×) into 49 mL of basal medium.
Medium Preparation
Since L-glutamine is unstable in aqueous solution and its degradation products are cytotoxic to cells, neither neuronal basal medium nor B-27 contains L-glutamine. Therefore, additional L-glutamine must be supplemented during medium preparation.
1. Thaw the product at 4 ℃.
2. Before use, aseptically add 2% of this product and 0.5 mM L-glutamine into neuronal basal medium to prepare complete neuronal medium.
(Note: The term "complete neuronal medium" hereinafter refers to neuronal basal medium supplemented with B-27 supplement and L-glutamine.)
3. Note: The remaining product may be aliquoted into working volumes and stored at -20 ℃. Thaw only the required volume for subsequent experiments. Avoid repeated freeze-thaw cycles.
4. For primary hippocampal neuron culture, supplement the prepared complete neuronal medium with an additional 25 μM L-glutamate. Do not add L-glutamate during medium replacement starting from day 4 of culture. The prepared complete medium can be stored protected from light at 2–8 ℃ for up to one week.
Cell Culture Procedure
The following protocol has been validated in primary cortical neurons from rat embryos.
1. Coat the culture surface (glass or cell-culture-grade plastic) with sterile cold 0.05 mg/L poly-L-lysine aqueous solution. Apply 0.15 mL per square centimeter, and incubate overnight (16 h) at 37 ℃.
2. Remove the poly-L-lysine solution and rinse the surface twice with sterile distilled water. Rinse thoroughly, as poly-L-lysine is cytotoxic to cells. Open the plate lid in a biosafety cabinet and allow air ventilation until all wells are completely dry. The dried plates can be used immediately or stored at 4 ℃ for a maximum of 2 weeks.
3. Isolate primary hippocampal/cortical neurons from rat embryos at embryonic day 16–18 according to laboratory standard operating procedures, or thaw cryopreserved primary rat neurons following the manufacturer’s instructions.
4. Pre-warm the complete neuronal medium in a 37 ℃ water bath. The recommended seeding density is 2×10⁵ cells per well (24-well plate format), which may be adjusted according to experimental requirements.
5. Place the seeded culture vessels in an incubator at 37 ℃ with 5% CO₂ for cultivation.
6. Replace half of the medium with fresh complete medium 16–24 hours after seeding; thereafter, perform medium change every 2–3 days.
Thawing and Culture of Cryopreserved Neurons:Important Notes:
1. Before thawing cells, rinse a sterile 15 mL conical tube with complete neuronal medium, then place it in a biosafety cabinet to air-dry.
2. When removing the cryovial from liquid nitrogen, slightly loosen the cap to release pressure, then tighten the cap again.
3. Gently swirl the cryovial in a 37 °C water bath to thaw cells rapidly within 2 minutes. Remove the vial when only a small amount of ice crystals remain inside (the vial still feels cold to the touch).
4. Tap the cryovial gently on the workbench to settle the liquid to the bottom. Use a 1 mL pipette tip pre-rinsed and dried with complete neuronal medium to transfer the cells extremely gently into the pre-rinsed and dried 15 mL conical tube.
5. Rinse the cryovial with 1 mL pre-warmed complete neuronal medium, then add the rinsing medium dropwise into the cell suspension in the 15 mL conical tube at a rate of one drop per second. Gently rotate the conical tube after each drop. Do not add all the medium into the tube at once.
6. Slowly add another 2 mL of pre-warmed complete neuronal medium dropwise to bring the total volume to 4 mL. Mix gently with a 1 mL pipette tip, taking care not to generate air bubbles.
7. Aspirate 10 μL of cell suspension with a pre-rinsed and dried pipette tip, add it into a microcentrifuge tube containing 10 μL of 0.4% trypan blue, and tap the tube gently to mix well. Determine viable cell density by manual cell counting. The viability of thawed cells should be higher than 50%.
8. Seed approximately 2×10⁵ cells per well into a 24-well plate pre-coated with poly-L-lysine (refer to Cell Culture Procedure), or seed at the desired cell density. Dilute the cell suspension with pre-warmed complete neuronal medium to a final volume of 500 μL per well. Culture the neurons according to Steps 5–6 in Cell Culture Procedure in an incubator at 37 °C with 5% CO₂
Important Notes:
1. Primary neurons can adhere directly to bare plastic or glass surfaces. It is recommended to pre-rinse the inner surface of all plastic and glassware with complete neuronal medium to maximize cell recovery and yield.
2. Cells are extremely fragile upon recovery from cryopreservation. Do not shake or centrifuge the cells throughout the entire operation.
3. It is recommended to thaw only one vial of cells at a time. Minimize the time required to transfer the cryovial from liquid nitrogen to a 37 °C water bath.
Precautions
1. Thaw the product overnight at 4 ℃ before use.
2. Mix the product thoroughly before addition when taking samples.
3. Perform strict aseptic operation during use to avoid microbial contamination.
4. For optimal performance, do not leave the product at room temperature or elevated temperature overnight.
5.Use the product within the shelf life. Discard any expired product.
ZJ26F0636933
危险品化学品经营许可证（带存储）