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10層細(xì)胞工廠

Human skin keratinocyte primary cultures can be established from skin biopsies with culture media containing epithelial growth factor (EGF). Although current methods are efficient, optimization is required to accelerate the procedure and obtain these cultures in less time. In the present study, we evaluated the effect of novel formulations based on EGF-loaded nanostructured lipid carriers (NLC). First, biosafety of NLC containing recombinant human EGF (NLC-rhEGF) was verified in immortalized skin keratinocytes and cornea epithelial cells, and in two epithelial cancer cell lines, by quantifying free DNA released to the culture medium. Then we established primary cell cultures of human skin keratinocytes with basal culture media (BM) and BM supplemented with NLC-rhEGF, liquid EGF (L-rhEGF), or NLC alone (NLC-blank). The results showed that cells isolated by enzymatic digestion and cultured with or without a feeder layer had a similar growth rate regardless of the medium used. However, the explant technique showed higher efficiency when NLC-rhEGF culture medium was used, compared to BM, L-rhEGF, or NLC-blank. Gene expression analysis showed that NLC-rhEGF was able to increase EGFR gene expression, along with that of other genes related to cytokeratins, cell–cell junctions, and keratinocyte maturation and differentiation. In summary, these results support the use of NLC-rhEGF to improve the efficiency of explant-based methods in the efficient generation of human keratinocyte primary cell cultures for tissue engineering use.

 
血清培養(yǎng)基瓶1000ml

人類皮膚角質(zhì)形成細(xì)胞原代培養(yǎng)物可以從皮膚活檢標(biāo)本中建立，培養(yǎng)基中含有上皮生長因子 (EGF)。雖然目前的方法是有效的，但需要優(yōu)化以加快程序并在更短的時間內(nèi)獲得這些文化。在本研究中，我們評估了基于裝載 EGF 的納米結(jié)構(gòu)脂質(zhì)載體 (NLC) 的新型制劑的效果。首先，通過量化釋放到培養(yǎng)基中的游離 DNA，在永生化皮膚角質(zhì)形成細(xì)胞和角膜上皮細(xì)胞以及兩種上皮癌細(xì)胞系中驗證了含有重組人 EGF (NLC-rhEGF) 的 NLC 的生物安全性。然后我們用基礎(chǔ)培養(yǎng)基 (BM) 和 BM 建立了人類皮膚角質(zhì)形成細(xì)胞的原代細(xì)胞培養(yǎng)物，其中補(bǔ)充了 NLC-rhEGF、液體 EGF (L-rhEGF) 或單獨的 NLC (NLC-空白)。結(jié)果表明，無論使用何種培養(yǎng)基，通過酶消化分離并在有或沒有飼養(yǎng)層的情況下培養(yǎng)的細(xì)胞都具有相似的生長速率。然而，與 BM、L-rhEGF 或 NLC 空白相比，當(dāng)使用 NLC-rhEGF 培養(yǎng)基時，外植體技術(shù)顯示出更高的效率?；虮磉_(dá)分析表明NLC-rhEGF能夠增加EGFR基因表達(dá)，以及與細(xì)胞角蛋白、細(xì)胞-細(xì)胞連接和角質(zhì)細(xì)胞成熟和分化相關(guān)的其他基因的表達(dá)?？傊?，這些結(jié)果支持使用 NLC-rhEGF 來提高基于外植體的方法在有效生成用于組織工程的人角質(zhì)形成細(xì)胞原代細(xì)胞培養(yǎng)物的效率。

For the biosafety analyses, immortalized human epithelial cells Ker-CT (CRL-4048), and corneal epithelial cells SIRC (ATCC CCL-60) were purchased from ATCC (Rockville, MD, USA) and cultured with the media recommended by the supplier. Cell lines corresponding to epidermoid carcinoma cells A431 (CRL-1555) and lung carcinoma cells A549 (CRM-CCL-185) were also obtained from ATCC and cultured as recommended.

Primary cell cultures of human skin keratinocytes (HKC) were established from small full-thickness skin biopsies obtained from healthy donors who underwent skin surgery. Immediately after excision, skin samples were kept at 4 °C in Dulbecco’s modified Eagle’s medium (DMEM; Merck, Darmstadt, Germany), supplemented with antibiotics and antimycotics (100 U/mL penicillin G, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B; Merck), and delivered to the laboratory. Biopsy samples were then washed twice in phosphate-buffered saline (PBS), surgically prepared to remove any remaining adipose tissue, and processed with two different methods routinely used to establish keratinocyte cultures:

(1)

Cell isolation by enzymatic digestion. Tissue samples were incubated in trypsin-EDTA as previously reported [24]. Briefly, samples were placed in a commercial solution containing 0.05% trypsin and 0.02% EDTA (Merck) at 37 °C with gentle shaking. After 20 min, the solution was harvested and inactivated with culture medium, and detached epithelial cells were harvested from the dissociation solution by centrifugation. This procedure was repeated up to 10 times, and all harvested cells were cultured together in 6-well plates. In one of the study groups, harvested cells were cultured on a feeder layer previously cultured on the plates. For this group, 3T3 cells (Merck) were cultured on 6-well plates and a lethal dose of gamma irradiation was applied when cells reached semiconfluence. The cells were washed with PBS, and then dissociated cells were cultured directly on the surface of this cell layer.

(2)

Explant technique. The tissues were trimmed into small pieces with a surgical blade, and each piece was placed on the surface of a 6-well plate, with the epithelial layer in direct contact with the culture surface. These explants were allowed to attach to the surface for 30 min, and a very small amount of culture medium was carefully added to prevent explant detachment. Culture plates were placed in a cell incubator, and 5 mL of culture medium was added to each well 24 h later.

In all cases, cells were incubated at 37 °C with 5% CO2 under standard culture conditions and with specific culture media in each study group (see Section 2.4). The culture medium was changed every 3 days.

 
T75細(xì)胞培養(yǎng)瓶

細(xì)胞培養(yǎng)

對于生物安全分析，永生化人上皮細(xì)胞 Ker-CT (CRL-4048) 和角膜上皮細(xì)胞 SIRC (ATCC CCL-60) 購自 ATCC (Rockville, MD, USA)，并使用供應(yīng)商推薦的培養(yǎng)基進(jìn)行培養(yǎng)。對應(yīng)于表皮樣癌細(xì)胞 A431 (CRL-1555) 和肺癌細(xì)胞 A549 (CRM-CCL-185) 的細(xì)胞系也從 ATCC 獲得并按推薦培養(yǎng)。

人類皮膚角質(zhì)形成細(xì)胞 (HKC) 的原代細(xì)胞培養(yǎng)物是從接受皮膚手術(shù)的健康供體獲得的小型全層皮膚活檢組織中建立的。切除后立即將皮膚樣品保存在 Dulbecco 改良 Eagle 培養(yǎng)基（DMEM；默克，德國達(dá)姆施塔特）中 4°C，輔以抗生素和抗真菌劑（100 U/mL 青霉素 G、100 mg/mL 鏈霉素和 0.25 mg/ mL 兩性霉素 B；Merck），并送到實驗室。然后將活檢樣本在磷酸鹽緩沖鹽水 (PBS) 中洗滌兩次，通過手術(shù)準(zhǔn)備去除任何剩余的脂肪組織，并使用兩種常規(guī)用于建立角質(zhì)形成細(xì)胞培養(yǎng)物的不同方法進(jìn)行處理：

(1)

通過酶消化分離細(xì)胞。如先前報道的那樣，組織樣品在胰蛋白酶-EDTA 中孵育 [ 24]。簡而言之，在 37°C 下，將樣品放入含有 0.05% 胰蛋白酶和 0.02% EDTA (Merck) 的商業(yè)溶液中，輕輕搖動。20分鐘后，收獲溶液并用培養(yǎng)基滅活，通過離心從解離溶液中收獲分離的上皮細(xì)胞。該過程重復(fù)多達(dá) 10 次，并將所有收獲的細(xì)胞一起培養(yǎng)在 6 孔板中。在其中一個研究組中，收獲的細(xì)胞在先前在平板上培養(yǎng)的飼養(yǎng)層上培養(yǎng)。對于該組，3T3 細(xì)胞（Merck）在 6 孔板上培養(yǎng)，當(dāng)細(xì)胞達(dá)到半融合時應(yīng)用致死劑量的 γ 輻射。用PBS洗滌細(xì)胞，然后將解離的細(xì)胞直接培養(yǎng)在該細(xì)胞層的表面上。

(2)

外植技術(shù)。用手術(shù)刀將組織剪成小片，每片置于6孔板表面，上皮層與培養(yǎng)面直接接觸。這些外植體被允許附著在表面 30 分鐘，并小心地加入非常少量的培養(yǎng)基以防止外植體脫離。將培養(yǎng)板置于細(xì)胞培養(yǎng)箱中，24 h后每孔加入5 mL培養(yǎng)基。

在所有情況下，細(xì)胞在 37 °C 下與 5% CO 2在標(biāo)準(zhǔn)培養(yǎng)條件下以及每個研究組中的特定培養(yǎng)基一起孵育。每3天更換一次培養(yǎng)基。

關(guān)鍵詞：表皮生長因子,納米粒子,國家圖書館,角質(zhì)形成細(xì)胞,組織工程,細(xì)胞培養(yǎng)
EGF, nanoparticles, NLC, keratinocytes, tissue engineering, cell culture

來源:MDPI https://www.mdpi.com/2227-9059/9/11/1634/htm