This work was supported by NIH grant EB007278.

Making a BFC
BFCs are made by molding polydimethylsiloxane (PDMS) over a 4" Si master wafer.
Making a master wafer
<ol>
<li>Start by dehydrating the Si wafers for 30 min at 130&deg;C. </li>
<li>Place the wafer on a spin-coater, pour SU8-2050 (Microchem) onto the wafer, ramp from 300 rpm/s to 3000-4000 rpm, and spin for 30 s to yield feature heights of 40-30 mm, respectively. </li>
<li>After spinning, place the wafer on a 65&deg;C hotplate, immediately ramp up the temperature to 95&deg;C for 5-6 min, and then ramp it down to 65&deg;C. </li>
<li>Expose the wafers to a UV dose of 200 mJ/cm2 on a contact aligner using a dark-field mask. </li>
<li>Place the wafers on a 65&deg;C hotplate, immediately ramp up the temperature to 95&deg;C for 4-5 min, and then ramp it down to 65&deg;C. </li>
<li>Develop the wafers in PM acetate and Isopropanol. </li>
<li>Silanize the wafers for 30 min using hexamethyldisiloxane (Shin-Etsu MicroSi), or (Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (t2492-KG, UCT Specialities, Bristol, PA), to prevent PDMS from adhering to the Si master wafer.</li>
</ol>
Molding chips
<ol>
<li>Mix PDMS (Dow Corning, Sylgard 184) in a 10:1 ratio, pour it over the master Si wafer (~15 g per wafer), and let it cure overnight at room temperature. </li>
<li>Peel the cured PDMS off the Si wafer and cut out each chip using a razor blade. </li>
<li>Bond each chip to a 1x1 cm cut microscope slide for easy handling.</li>
</ol>
Preparing the BFC for use
<ol>
<li>Soak the BFC overnight in PBS in order to prevent absorption of media into the PDMS during the experiment. </li>
<li>Dry the chips with a Kimwipe (Kimtech Science). </li>
<li>Put a drop of 7.5% Bovine Serum Albumin (BSA) (~200 mL) on the patterned surface of the BFC, and then scrape it with a pipette tip to disperse the BSA and remove any bubbles from the wells. </li>
<li>Leave the BSA on the BFC surface for at least 0.5 hours. Ideally agitate the BSA every 15 min to prevent crusting. </li>
<li>To fit the BFC inside a 35x10 mm TCPS dish (Falcon, 35-3001), cut the rims of a TCPS dish half way down so that the &frac34;" binder clips (Office Depot) will fit over the dish rim to clamp the chip and dish together. </li>
<li>Cut a spacer gasket (frame-shaped with 20&acute;20 mm outer edge, 15&acute;15 mm inner edge, and 250-500 mm thickness) from a PDMS sheet (Silicone Specialty Products) or any other silicone, and apply it to the dish using tweezers.</li>
</ol>
Patterning cells with a BFC
<ol>
<li>Sterilize the dish, gasket, BSA-coated chip, and 2 binder clips under UV light for 1 minute. </li>
<li>Aspirate the BSA and rinse the BFC twice with 200 mL PBS. </li>
<li>Add gelatin to the cut TCPS dish if required for your cell-line. Otherwise, wet the TCPS surface with some media before applying it to the chip (see below). This prevents bubbles from forming in the chamber. </li>
<li>After aspirating the PBS, pipette 200 mL of cell solution (~1&times;106 cells/mL) onto the BFC surface. Let the cells settle for 5-10 min. </li>
<li>Tilt the BFC to one corner at a ~15&deg; angle and slowly pipette the cell solution off with a 200 mL pipette. Then, place the BFC flat and add 100 mL of PBS or blank media to the opposite BFC corner. Rinse the BFC in this manner an additional 2-4 times, as necessary, to clear all cells from the inter-well regions. </li>
<li>Pipette 200 mL of media onto the chip surface. Aspirate gelatin/media from the TCPS. Then invert the pre-wetted dish and slowly push the BFC up, into the dish. </li>
<li>Apply a binder clip each, to two sides of the chamber to seal it. Remove the metal prongs from the binder clips so that dish remains level when flipped over. </li>
<li>Finally, quickly flip the setup over while avoiding any unnecessary movement, and place it in the incubator.</li>
</ol>

<ol>
	<li>Rosenthal, A., Macdonald, A., Voldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Patterning+Chip+for+Controlling+the+Stem+Cell+Microenvironment.">Cell Patterning Chip for Controlling the Stem Cell Microenvironment.</a> Biomaterials. 28, 3208-3216 (2007).</li></ol>

Although the protocol and video shown here describe using the BFC 1 to pattern mouse embryonic stem cells (mESCs), BFCs can be used to pattern practically any cell type of interest. The only change one may need to make is to alter the size of the microwells. In our experience, to use the BFC to pattern single cells of another cell type, a microwell diameter and height equal to 10 microns greater than the unattached cell diameter works best.
BFCs can also be used to pattern cells on...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dw Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Silicon master mould</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>35-3001</td>
<td>35 mm</td>
</tr>
<tr>
<td>3/4" binder clips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>hexamethyldisiloxane</td>
<td>&nbsp;</td>
<td>Shin-Etsu MicroSi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>New Item</td>
<td>&nbsp;</td>
<td>Microchem</td>
<td>SU8-2050</td>
<td>&nbsp;</td>
<tr>
<td>PM acetate</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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patterning of embryonic stem cells using the bio flip chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

Watch this Scientific Journal Video about Patterning of Embryonic Stem Cells Using the Bio Flip Chip at JoVE.com

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

patterning-of-embryonic-stem-cells-using-the-bio-flip-chip

Research

JoVE Journal

Biology

9.4K Views.  MIT - Massachusetts Institute of Technology. We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Video: Patterning of Embryonic Stem Cells Using the Bio Flip Chip

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.

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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.

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 ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...