HELIOS 人血小板裂解物-PURE（GMP級(jí)）
 

四、質(zhì)量控制測(cè)試
外觀(guān)： 黃色至淺黃色液體。
總蛋白： 4.0 - 8.0 g/dL。
pH值： 6.5 - 8.5。
滲透壓： 275 - 355 mOsm/Kg。
內(nèi)毒素: ≤ 10 EU/mL
支原體： 未檢測(cè)到
無(wú)菌性： 無(wú)生長(zhǎng)
性能測(cè)試： 測(cè)試和記錄
纖維蛋白原： 20微克/毫升

五、應(yīng)用范圍
用于人類(lèi)體外組織和細(xì)胞培養(yǎng)加工應(yīng)用

六、重要信息
1. 不溶性顆粒可能會(huì)在解凍的 UltraGRO 中形成™-純正。已發(fā)表的研究表明，顆粒不會(huì)改變產(chǎn)品的性能。
2.不溶性顆粒可能會(huì)在解凍的 UltraGRO 中形成™-PURE，建議在3,400 xg下離心3~5分鐘。
3.在 UltraGRO 之后過(guò)濾完成介質(zhì)（例如 5%）™-PURE在基礎(chǔ)介質(zhì)中稀釋?zhuān)粫?huì)影響UltraGRO™-PURE補(bǔ)充細(xì)胞培養(yǎng)性能。然而，0.22微米過(guò)濾 不 推薦用于100% UltraGRO™-純凈濃縮物，因?yàn)檫@可能會(huì)減少5%的UltraGRO™-純細(xì)胞培養(yǎng)性能
4.應(yīng)避免反復(fù)的凍融循環(huán)，因?yàn)樗鼈儠?huì)導(dǎo)致不可溶性沉淀物的增加，從而導(dǎo)致 UltraGRO 的潛在減少™-純粹的表演。

七、MSC培養(yǎng)條件
1.媒體：完全培養(yǎng)基由基礎(chǔ)培養(yǎng)基(例如α-MEM或其他支持性培養(yǎng)基)和UltraGRO-PURE組成。
2.培養(yǎng)類(lèi)型：粘附
3.培養(yǎng)容器：細(xì)胞培養(yǎng)板、T型瓶、G-Rex培養(yǎng)瓶、細(xì)胞培養(yǎng)袋、旋轉(zhuǎn)瓶或垂直輪式生物反應(yīng)器溫度范圍：36℃至38℃
4.培養(yǎng)箱環(huán)境：濕度為4~6%的CO加濕環(huán)境，確保培養(yǎng)容器中實(shí)現(xiàn)適當(dāng)?shù)臍怏w交換。

八、使用說(shuō)明
1. UltraGRO™-PURE在典型的細(xì)胞培養(yǎng)介質(zhì)中，即含有2mM L-谷氨酰胺的最終濃縮物的α-MEM中，以5%（v/v）的濃度顯示MSC的******生長(zhǎng)。
2. 我們建議將MSCs的接種量控制在每平方厘米約3×103~ 6×103個(gè)細(xì)胞。
3. UltraGRO™-PURE已經(jīng)被纖維蛋白原耗盡，并且在細(xì)胞培養(yǎng)液中不需要添加肝素。

九、使用詳細(xì)說(shuō)明
1. 產(chǎn)品準(zhǔn)備
解凍：將產(chǎn)品從-20℃冰箱取出，于2-8℃冰箱中緩慢解凍（建議過(guò)夜），或37℃水浴快速解凍（需輕輕混勻，避免泡沫產(chǎn)生）。解凍后輕輕顛倒混勻，肉眼觀(guān)察無(wú)沉淀或絮狀物即可使用。
分裝：為避免反復(fù)凍融，建議在無(wú)菌條件下將解凍后的裂解物分裝至無(wú)菌離心管中，每管體積根據(jù)單次用量確定（如10 mL/管），分裝后立即放回-20℃保存。
2. 培養(yǎng)基配制
基礎(chǔ)培養(yǎng)基選擇：根據(jù)目標(biāo)細(xì)胞類(lèi)型選擇無(wú)血清基礎(chǔ)培養(yǎng)基（如MSC專(zhuān)用無(wú)血清培養(yǎng)基、 endothelial cell basal medium等）。
添加比例：常規(guī)培養(yǎng)推薦添加體積分?jǐn)?shù)5%-10%的HELIOS人血小板裂解物-PURE（具體比例需根據(jù)細(xì)胞類(lèi)型優(yōu)化，如MSC建議5%-8%，內(nèi)皮細(xì)胞建議8%-10%）。
配制方法：在無(wú)菌操作臺(tái)中，將預(yù)熱至室溫的基礎(chǔ)培養(yǎng)基與血小板裂解物按比例混合，輕輕混勻，避免劇烈震蕩產(chǎn)生泡沫。配制完成后可立即使用，或2-8℃保存不超過(guò)7天。
3. 細(xì)胞培養(yǎng)操作
細(xì)胞復(fù)蘇與接種：按常規(guī)細(xì)胞復(fù)蘇流程操作，復(fù)蘇后的細(xì)胞以適宜密度接種至含HELIOS血小板裂解物的培養(yǎng)基中（如MSC接種密度1×10?-5×10? cells/cm²），置于37℃、5% CO?培養(yǎng)箱中培養(yǎng)。
換液頻率：根據(jù)細(xì)胞生長(zhǎng)速度調(diào)整，通常每2-3天換液一次。換液時(shí)棄去舊培養(yǎng)基，用PBS輕柔洗滌細(xì)胞1-2次，加入新鮮配制的培養(yǎng)基。
細(xì)胞傳代：當(dāng)細(xì)胞融合度達(dá)到70%-80%時(shí)進(jìn)行傳代，棄去培養(yǎng)基后用胰酶（如0.25% Trypsin-EDTA）消化，終止消化后離心收集細(xì)胞，按1:3-1:5比例接種至新培養(yǎng)器皿中。
凍存與復(fù)蘇：細(xì)胞凍存時(shí)，可在凍存液中添加10%-20%的HELIOS血小板裂解物（替代FBS），提高凍存細(xì)胞活性；復(fù)蘇時(shí)按常規(guī)流程操作，使用含血小板裂解物的培養(yǎng)基重懸細(xì)胞。
4. 注意事項(xiàng)
避免反復(fù)凍融：反復(fù)凍融會(huì)導(dǎo)致生長(zhǎng)因子活性降低，建議分裝后單次使用。
無(wú)菌操作：全程嚴(yán)格無(wú)菌操作，避免污染；培養(yǎng)基配制后若出現(xiàn)渾濁、沉淀或顏色異常，禁止使用。
濃度優(yōu)化：不同細(xì)胞系對(duì)血小板裂解物的敏感性存在差異，初次使用建議通過(guò)梯度實(shí)驗(yàn)（如2%、5%、10%、15%）確定******添加比例。
兼容性：與多數(shù)無(wú)血清基礎(chǔ)培養(yǎng)基兼容，若需與其他添加劑（如生長(zhǎng)因子、抗生素）聯(lián)合使用，建議先進(jìn)行兼容性測(cè)試。
安全提示：產(chǎn)品雖經(jīng)嚴(yán)格病毒滅活處理，但仍需按生物安全二級(jí)（BSL-2）標(biāo)準(zhǔn)操作，避免直接接觸皮膚或黏膜。
5. 常見(jiàn)問(wèn)題解決
細(xì)胞生長(zhǎng)緩慢：檢查添加比例是否過(guò)低，可嘗試提高至10%；或確認(rèn)細(xì)胞接種密度是否適宜，調(diào)整接種濃度。
細(xì)胞貼壁不良：可能因血小板裂解物中黏附蛋白含量不足，可短暫提高添加比例至10%-15%，或在培養(yǎng)基中額外添加纖連蛋白（1-5 μg/mL）。
培養(yǎng)基渾濁：提示可能污染，立即終止培養(yǎng)并檢測(cè)污染源，嚴(yán)格無(wú)菌操作重新配制培養(yǎng)基。

十、儲(chǔ)存和運(yùn)輸
UltraGRO™-PURE在需要之前冷凍儲(chǔ)存最穩(wěn)定。建議儲(chǔ)存溫度為-20°C。濕冷凍UltraGRO™-使用前將產(chǎn)品置于37°C水浴中純凈。一次UltraGRO™-PURE解凍后，建議當(dāng)天完全使用完成的培養(yǎng)基制備（例如5%），或?qū)⑵浞殖梢淮涡杂昧?，并?20°C下儲(chǔ)存未使用的用量。

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