这项工作得到了山东省自然科学基金重点项目（ZR2019ZD36）和山东省重点研究发展计划（2019GSF108107）的支持。山东省重点研发计划（2018GSF118240）向J.G.：山东省医疗卫生科技发展项目（2018WS163）至Z.X.，山东省医疗卫生科技发展项目（2019WS045）至J.S.

根据该学会人类研究伦理委员会的指导方针，本协议中使用的人体组织是从Maxillo面部外科拔出受影响的牙齿中丢弃的新鲜成人金银杏组织。GR201711，日期：02-27-2017）。
1. 准备工作
<ol><li>在含有 10 mL 磷酸盐缓冲盐水 （PBS） 的 15 mL 管中收集新鲜的成人金银杏组织，辅以 3% 青霉素/链霉素 （P/S），并将组织保持在 4 °C。 注：切除后24小时内将下面的组织详细处理。</li>
</ol>2. 准备试剂和文化媒介
<ol><li>准备洗涤溶液：3% P/S. 将 50 mL 的 PBS 与 1.5 mL 的青霉素 （100 U/mL） 和 1.5 mL 链霉素 （100 毫克/升） 混合。</li>
	<li>准备500 mL的生长因子含生长因子介质（称为G-中型）：DMEM/F12（3：1）介质，含1%P/S，2%B27补充剂，20 ng/mL表皮生长因子（EGF），40 ng/mL成纤维细胞生长因子2（FGF2）和40微克/mL真菌酮。</li>
	<li>为新方法准备酶消化溶液：在 50 mL 的 DMEM 中溶解 125 毫克的脱脂粉和 125 毫克的 I 型拼贴粉。 注意：通过 0.22 μm 过滤器过滤所有酶溶液，并在 4 °C 下存储。</li>
	<li>准备500 mL的介质中和酶消化：10%的胎儿牛血清（FBS）和1%的P/S在DMEM介质。</li>
</ol>3. 直接外植方法
<ol><li>金吉瓦尔组织预处理<ol>
<li>用 5 mL 的 75% 乙醇清洗金银组织 30s 。然后，用 5 mL 的洗涤液（步骤 2.1）冲洗两次，5 分钟。 注：在直径为50毫米的细胞培养皿中进行所有洗涤。</li>
	</ol>
</li>
	<li>金吉瓦尔上皮细胞培养<ol>
<li>将金银组织切成1毫米3 片，撒在100毫米细胞培养皿中。使用精细的尖钳和眼科剪刀进行切割操作。</li>
		<li>将 G 中等（步骤 2.2）添加到培养皿中。然后，在 37 °C 的 5% CO2 孵化器中孵育培养皿。 使用倒置显微镜（40倍）每24小时检查一次组织。</li>
		<li>当上皮细胞在组织片周围传播到直径为 2-5 mm 时，取出金银杏组织片。每 2-4 天更改一次 G-中型。</li>
	</ol>
</li>
</ol>4. 新的改性酶方法
<ol><li>金吉瓦尔组织预处理<ol>
<li>为此，请遵循与描述的直接除植物方法相同的步骤，步骤 3.1.1。</li>
	</ol>
</li>
	<li>金银组织消化<ol>
<li>用两个消毒刀片将金银组织切成小块，大小约为<1毫米。用两个手术刀片重复粉碎过程。</li>
		<li>将 1 mL 2.5 毫克/mL 脱脂 ® 拼贴剂溶液添加到包含金银组织碎片的 1.5 mL 离心机管中，然后在 37 °C 的孵化器中孵育管 15-20 分钟。</li>
		<li>当组织变得透明和絮落时，加入1 mL的中和溶液来终止消化。通过上下管道 10-15 次彻底混合溶液。通过 100 μm 网状过滤器传递解决方案。</li>
		<li>离心机在 200 x g 5 分钟。</li>
		<li>取出超高颗粒，在含有生长因子的介质（步骤 2.2）的 10 mL 中将细胞颗粒重新悬浮在底部。将细胞悬浮转移到 100 mm 细胞培养盘中。在细胞培养皿中加入 10 μM Y-27632。</li>
		<li>在 5% CO2 孵化器中培养 37 °C 的细胞。每 2 天用新鲜的 G 媒体替换旧的 G 媒体。在第 1 天和第 3 天观察细胞。</li>
	</ol>
</li>
</ol>5. 细胞传递
<ol><li>将盘子从孵化器中取出，取出用过的介质，用 PBS 洗两次。每100毫米菜加入2mL的0.05%试用素。 注意：摇动盘，以确保 Trypsin 溶液和菜底之间有足够的接触。</li>
	<li>将盘子放在孵化器中约 5 分钟，在 37 °C 处进行消化过程。</li>
	<li>使用显微镜 （40x） 检查细胞，并确保大多数细胞与盘底分离。</li>
	<li>加入2mL的中和溶液，以阻止消化，并将细胞转移到15mL管中。派佩特上下 10 - 15 次。将细胞以 200 x g 为离心，5 分钟。</li>
	<li>慢慢取出超高超。用10mL的G-中等重用细胞，并计算细胞数量。</li>
	<li>在 10 mL的 G 中型和 10 μM Y-27632 中加入约 1 x 10 6 个细胞到每个 100 mm 盘中。</li>
	<li>每 2 天更新一次 G 中型和 Y-27632。查看第 1 天和第 5 天的单元格。</li>
</ol>

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牙周组织是维持牙周完整性和健康的关键结构。金吉瓦尔上皮细胞在牙周组织的修复和再生中起着重要作用，可用于科学研究和临床应用及相关领域，包括口腔生物学、药理学、毒理学和口腔粘膜缺乏症18。因此，有必要开发一种稳定而有效的方法来收获口腔上皮细胞19。原发性上皮细胞是一种完全分化的细胞，通道少，寿命短。培育上皮细胞已被证明是比培养...

人牙石是保护牙周组织的第一道防线结构，它不仅是物理和化学屏障1，而且分泌不同类别的炎症调解员参与免疫反应，构成免疫屏障2、3。金银上皮在牙周组织的修复和再生中起着重要的作用。因此，研究牙周炎的防御和免疫，对于了解牙周炎的发生、诊断和治疗具有重要意义。从人类金银杏组织中分离和培养金吉瓦尔上皮细胞是研究金吉瓦尔上皮的第一步。这种程序需要基本操作，如为组织工程生产种子细胞、牙周相关疾病的体外模型以及修复牙周缺陷的材料。
原发性金银体上皮细胞的特点是体外4的分化率低，几十年来，研究人员一直在寻找最佳的分离和培养方法。迄今为止，实验室中通常使用两种不同的技术，即直接外生法和酶法，在体外获得原发性金吉瓦尔上皮细胞。直接除种方法具有组织标本量低、隔离程序简单等优点，但具有培养时间长、易受污染等优点。虽然酶方法缩短了所需的培养时间，但效率相对较低，并且因使用的酶和介质而异。Kedjarune等人6表明，与酶方法相比，直接外植方法在亚文化（2周）之前需要更多的时间，在培养金吉瓦尔上皮细胞方面似乎更为成功。然而，比较这两种方法，Klingbeil等人发现，这种酶方法对口腔上皮细胞的原发培养效果最好，并且有可能在最短的时间内（11.9天对14.2天）获得最佳细胞产量。
因此，开发一种更方便、更有效的口腔上皮细胞分离与培养方法非常重要。我们之前报告说，添加Y-27632，Rho相关蛋白激酶（ROCK）的抑制剂，简化了人类原发性表皮细胞和角蛋白细胞从成人皮肤组织8，9，10的分离程序。我们开发了G-media，一种新的条件接种介质，自发地将表皮与皮肤细胞分离，支持原发性表皮细胞8、9、10的生长和产量。在本研究中，我们通过将G-中型细胞与Y-27632结合，开发出一种新的无血清分离培养技术。从本质上讲，我们的方法是基于对传统两步酶方法的简化，因此我们比较了新方法和直接外生方法。这种改良的酶方法大大缩短了将金吉瓦尔上皮细胞与金银杏组织分离所需的时间，并提高了培养金吉瓦尔上皮细胞的效率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Names</td>
			<td></td>
			<td></td>
			<td>Abbreviations &#38; Comments</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Shanghai Ruiyu Bio-science&#38;Technology Co.Ltd.</td>
			<td>BBA0218AC</td>
			<td>Automatic cell counting</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubation</td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>50 ml Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifugation</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge Tubes</td>
			<td>KIRGEN</td>
			<td>190691J</td>
			<td>For cell digestion</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning incorporated</td>
			<td>431792</td>
			<td>Cell filtration</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Solarbio Life Science</td>
			<td>P1020-500</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>C11995500</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Defined K-SFM</td>
			<td>Life Technologies</td>
			<td>10785-012</td>
			<td>Gingival epithelial&#160; cells culture medium</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1AC5</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td>For HGGEPCs dissociation</td>
		</tr>
		<tr>
			<td>Dilution Medium</td>
			<td>Life Technologies</td>
			<td>50-9701</td>
			<td>For coating matrix</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For HGGEPCs isolation</td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Life Technologies</td>
			<td>17100-017</td>
			<td>For HGGEPCs isolation</td>
		</tr>
		<tr>
			<td>F12 Nutrient Mix, Hams</td>
			<td>Life Technologies</td>
			<td>31765035</td>
			<td>Component of G-medium</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr>
			<td>FGF-2 Millipore</td>
			<td>Merck Biosciences</td>
			<td>341595</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Gene Operation</td>
			<td>IAD1011</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Gibco</td>
			<td>15290026</td>
			<td>Preparation for G-medium</td>
		</tr>
		<tr>
			<td>EGF Recombinant Human Protein</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>Growth factor in G-medium</td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8</td>
			<td>Dojindo Molecular Technologies</td>
			<td>CK04</td>
			<td>For Cell proliferation assay</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human CK18</td>
			<td>Abcam</td>
			<td>ab82254</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human Cytokeratin10</td>
			<td>Abcam</td>
			<td>ab76318</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>Mouse anti-human Vimentin</td>
			<td>Cell Signaling Technology</td>
			<td>3390</td>
			<td>For immunofluorescence staining of Gingival fibroblasts</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human pan-ck</td>
			<td>BD</td>
			<td>550951</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-Ki67</td>
			<td>Abcam</td>
			<td>15580</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-p63</td>
			<td>Biolegend</td>
			<td>619002</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-p75NGFR</td>
			<td>Abcam</td>
			<td>ab52987</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary human gingival epithelial cells using y-27632

牙周组织是保护牙周组织的第一个结构，在许多口腔功能中起着有意义的作用。金银核酸上皮是金银组织的重要结构，特别是在牙周组织的修复和再生中。研究金吉瓦尔上皮细胞的功能具有重要的科学价值，如修复口腔缺陷和检测生物材料的兼容性。由于人类上皮细胞是高度分化的角蛋白细胞，其寿命短，难以通过。到目前为止，只有两种方法可以分离和培养金吉瓦尔上皮细胞，一种是直接的外生方法，另一种是酶。然而，使用直接外生法获得上皮细胞所需的时间较长，酶法的细胞存活率较低。临床上，金银组织采集有限，因此需要一个稳定、高效、简单的 体外 隔离培养系统。我们通过添加Y-27632（一种与Rho相关的激酶（ROCK）抑制剂，改进了传统的酶方法，这种抑制剂可以选择性地促进上皮细胞的生长。我们改良的酶法简化了传统酶方法的步数，提高了上皮细胞的培养效率，比直接除菌法和酶法具有显著优势。

图1 显示了直接外分法和改性酶法的示意图。在整个过程中，直接外生方法不需要任何消化酶。相比之下，传统的酶方法通常需要两套消化酶，脱口而出和拼贴，将上皮板从底层成纤维细胞层分离，然后尝试将上皮细胞释放到悬浮中。我们的新方法省略了分离的步骤，是一种简化的酶方法。此外，在G-中型中加入Y-27632可有效促进上皮细胞生长。直接外植方法通常需要大约2周?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632 at JoVE.com

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

在这里，我们提出了一个改进的方法，通过添加岩石抑制剂，Y-27632，以传统的方法隔离和培养人类金吉瓦尔上皮细胞。这种方法更容易，更省时，提高干细胞的特性，并产生更多的高潜力上皮细胞，无论是为实验室和临床应用。

使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

因此，这种基于细胞的协议的总目标是分离和遇到人类金吉瓦尔表皮细胞，以改善直接茄子方法的缺点。从新的改良内分泌方法，我们可以在相对较短的时间内获得更多的原发性金银核素上皮细胞，基于我们的材料和设备。因此，根据该研究所人类研究伦理委员会的指南，本协议中使用的人体组织是从Maxillo面部外科拔出的受撞击牙齿中丢弃的新鲜成人金银花组织。然后，用洗涤液两次，每次五分钟。金吉瓦尔组织的消化。 用两个手术刀片把组织切成小块。将 2.5 毫克/毫利特脱口而起和拼贴剂-1 溶液添加到 1.5 英里离心机管中。将组织件放入 1.5 英里离心机管中。在 37 分钟内孵育 15-20 分钟

isolation-culture-primary-human-gingival-epithelial-cells-using-y

Research

JoVE Journal

Bioengineering

4.7K 次观看.  Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration. 因此，这种基于细胞的协议的总目标是分离和遇到人类金吉瓦尔表皮细胞，以改善直接茄子方法的缺点。从新的改良内分泌方法，我们可以在相对较短的时间内获得更多的原发性金银核素上皮细胞，基于我们的材料和设备。因此，根据该研究所人类研究伦理委员会的指南，本协议中使用的人体组织是从Maxillo面部外科拔出的受撞击牙齿中丢弃的新鲜成人金银花组织。然后，用?...

视频: Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

科研

生物工程

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

使用光声血流循环黑色素瘤细胞的检测和隔离

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

人原发性前列腺组织培养的处理与评价

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...