<ol><li>一个健康，成倍扩大培养，用PBS洗，以去除残留的媒体，否则可能淬火胰蛋白酶反应。 </li><li>回暖的0.05％胰蛋白酶EDTA，然后添加到覆盖细胞，板允许在室温下5分钟孵化。在这段时间内可以观察细胞四舍五入，和殖民地抬离板面。 </li><li>温和的反复吹打将删除从钢板表面的细胞和殖民地。 </li><li>放置在一个标准的锥形管，包含预热的胚胎干细胞媒体淬火剩下的胰蛋白酶，胰酶消化细胞，然后纺制。 </li><li>细胞悬浮在大约一百万个细胞的浓度在媒体中的生长介质。 </li><li>充分重悬细胞后，冰冷的冻结媒体在相同体积的增加。 </li><li>细胞悬浮液充分混合，分装成冷冻小瓶，允许慢慢冻结超过24小时- 80C。 </li><li>冷冻细胞可以长期贮存的液氮转移到气相，或约6个月保持在-80。 </li></ol>注：在106细胞/ ml的浓度冷冻等分足够的复活一种文化上10厘米板。

<ol>
	<li>Cowan, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+Embryonic+Stem+Cells+from+Human+Blastocysts.">Derivation of Embryonic Stem Cells from Human Blastocysts.</a> , 1650-1656 (2004).</li></ol>

冻结色调细胞株中描述的技术，我们有效地存储和复活的色调细胞株和利益的任何哺乳动物细胞。工作时，与胚胎干细胞系，染色体核型异常，可能会出现不正常的细胞可以迅速接管该行，冷冻的细胞在每一个通道的一小部分是一个重要的考虑因素。因此，有冷冻分割在某些段落中除了大银行，在每一个通道是非常可取的。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HuES Medium</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For aq total of 651.5 ml, add: 500 ml KO-DMEM + 6.5 ml PenStrep + 6.5 ml L- Glut + 6.5 ml NEAA 6.5 ml + beta-mercaptoethanol 0.6ul (14.3 M stock) + 65 ml Serum Replacement + 65 ml Plasmanate + 0.65 ml bFGF2 (~10 ng/mL final)
</td>
</tr>
<tr>
<td>Freezing Medium</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>80% FCS (fetal calf serum) + 20% DMSO (dimethyl sulphoxide). Stored at 4C. </td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

freezing human es cells

在这里，我们证明了我们的实验室如何冻结色调人类胚胎干细胞系。一个健康，成倍扩大培养，用PBS洗，以去除残留的媒体，否则可能淬火胰蛋白酶反应。回暖的0.05％胰蛋白酶EDTA，然后添加到覆盖细胞，板允许在室温下5分钟孵化。在这段时间内可以观察细胞四舍五入，和殖民地抬离板面。温和的反复吹打将删除从钢板表面的细胞和殖民地。放置在一个标准的锥形管，包含预热的胚胎干细胞媒体淬火剩下的胰蛋白酶，胰酶消化细胞，然后纺制。细胞悬浮在大约一百万个细胞的浓度生长介质，在一个媒体毫升，浓度等，一个冻结等分足够的复活一种文化上10厘米板。充分重悬细胞后，冰冷的冻结媒体是在同等体积增加。细胞悬浮液充分混合，分装成冷冻小瓶，允许慢慢冻结超过24小时- 80C。冷冻细胞可以长期贮存的液氮转移到气相，或约6个月保持在-80。

Watch this Scientific Journal Video about Freezing Human ES Cells at JoVE.com

Freezing Human ES Cells

在这里，我们证明了我们的实验室如何冻结色调人类胚胎干细胞系。

冻结人类胚胎干细胞

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Research

JoVE Journal

Biology

14.9K 次观看.  Harvard. 在这里，我们证明了我们的实验室如何冻结色调人类胚胎干细胞系。

视频: Freezing Human ES Cells

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

科研

生物学

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

闪存冻结和Cryosectioning E12.5小鼠脑

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

冷冻，解冻，和运输包装细胞

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

修改ES / OP9共培养协议，提供增强造血后代的表征

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...