We deeply appreciate Dr. Takeshi Ikeya and technical staff&#160;in Toyo Gosei, Tokyo, Japan for providing thermosensitive micropatterned culture plates as well as scientific advice. We also thank Ms. Satomi Ogura, Ms. Sae Suzuki,&#160;Ms. Asuka Miyoshi and Ms. Katsue Morii for technical assistance with animal experiments. This work was financially supported in part by the JSPS KAKENHI Grant-in-Aid for Scientific Research, the Center of Innovation (COI) Program and the S- innovation program from the Japan Science and Technology Agency (JST), and the JSPS Core-to-Core Program, A. Advanced Research Networks.

All the animal studies were conducted with the approval of the Animal Care and Use Committee of the University of Tokyo, Tokyo, Japan.
1. Cell Preparation
<ol>
	<li>For primary hepatocytes, follow the protocol for hepatocyte isolation from rat by a modified two-step collagenase digestion process13,14.
	<ol>
		<li>Anesthetize Sprague Dawley (SD) rats (male, 5 weeks old) under inhalation anesthesia with isoflurane. Place a rat in a chamber connected to an anesthesia machine to provide isoflurane for the chamber. Take out the rat after falling asleep, and set it on the operating table with ventilation using a mask. Control the isoflurane flow approximately at 0.4 &#8211; 0.7 L/min by checking the conditions of the rat.&#160;Perfuse the livers of Sprague Dawley (SD) rats (male, 5 weeks old) from the hepatic portal vein with a special solution composed of 8 g/L sodium chloride (NaCl), 400 mg/L potassium chloride (KCl), 78 mg/L sodium dihydrogen phosphate dihydrate (NaH2PO4&#183;2H2O), 151 mg/L disodium hydrogen phosphate dodecahydrate (Na2HPO4&#183;12H2O), 2.38 g/L 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES), 190 mg/L ethylene glycol tetraacetic acid (EGTA), 350 mg/L sodium hydrogen carbonate (NaHCO3), and 900 mg/L glucose.</li>
		<li>Circulate collagenase solution through the liver. &#160; 
		NOTE: The solution is composed of 500 mg/L collagenase, 9.8 g/L Hank&#8217;s buffered salt, 2.38 g/ml HEPES, 556 mg/ml calcium chloride hydrate (CaCl2&#183;H2O), 350 mg/L NaHCO3, and 50 mg/L trypsin inhibitor, with the pH adjusted to 7.2.</li>
		<li>Remove the liver carefully, and mince it gently on a dish using a scalpel blade, add Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and filter the cell suspension through a 100 &#956;m nylon mesh. To remove additional debris, centrifuge the cell suspension at 20 &#215; g for 1 min. At this step, hepatocytes are in the supernatant.</li>
		<li>Repeat the centrifugation step twice (a total of three centrifugations). Finally, centrifuge the hepatocytes at 50 &#215; g for 3 min to recover them in the form of a pellet.</li>
		<li>Re-suspend the hepatocytes to a concentration of 4 &#215; 105 cells/ml in a special culture medium composed of DMEM&#160;supplemented with 10%&#160;FBS, 1% Pen-Strep-Glut (PSQ), 1% dimethyl sulfoxide (DMSO), 0.1 &#181;mol/L dexamethasone, 0.5 &#181;g/ml insulin, 10 mmol/L nicotinamide, 0.2 mmol/L phosphorylated ascorbate (Asc-2P), and 10 ng/ml human epidermal growth factor (hEGF)15. This special medium is mandatory for preserving the hepatic function under in vitro conditions.</li>
	</ol>
	</li>
	<li>For obtaining rat MSCs, euthanize Sprague Dawley (SD) rats (male, 5 weeks old) by excessive administration of isoflurane. Resect the femurs and tibias, and collect the bone marrows by inserting a 22 G needle into the shaft of the bone to flush it out with 10 ml of DMEM&#160;supplemented with 10%&#160;FBS. Collect the cells by filtration through a 100 &#181;m nylon mesh.</li>
	<li>Seed the cells onto 10 cm culture dishes using DMEM containing 10% FBS and 1% penicillin&#160;/&#160;streptomycin. For the spheroid experiments, use MSCs within 5 passages.</li>
</ol>
2. Preparation of 3D Cell Spheroids
<ol>
	<li>Commercially obtain micropatterned culture plates, on which the cell adhesive areas are regularly arrayed at 100 &#181;m diameter in a two-dimensional manner, surrounded by non-adhesive areas coated by the PEG matrix. &#160; 
	NOTE: For cell transplantation, an additional coating with the thermosensitive polymer PIPAAm is necessary to allow cell detachment by cooling the plates (see step 5.1). Temperature fluctuations can induce changes in the chemistry of this polymer8-10. At 37 &#176;C, PIPAAm is slightly hydrophobic, allowing the cells to be cultured under normal conditions. A decrease in the temperature below 32 &#176;C results in rapid hydration of the polymer, leading to the spontaneous detachment of the cells.</li>
	<li>Seed the hepatocytes or MSCs on 12-well micropatterned plates at a density of 4 &#215; 105 cells/well andincubate them at 37 &#176;C in a humidified atmosphere containing 5% CO2. The cells will accumulate on the micropatterned adhesion areas and gradually form round spheroids in 2 days. &#160; 
	NOTE: For preparing control cells in a monolayer culture, use normal 12-well plates and seed the cells at an identical density, following a similar procedure as described above.</li>
</ol>
3. Preparation of Polyplex Nanomicelles
<ol>
	<li>Synthesize a block copolymer, PEG-PAsp(DET) (poly[N&#8217;-[N-(2-aminoethyl)-2-aminoethyl] aspartamide]) (PEG Mw = 12,000, polymerization degree (DP) of PAsp(DET) segment = 59), and a homopolymer that is composed of only the cationic segment [PAsp(DET)] (DP = 55), following the procedures previously described by the authors11,16,17.</li>
	<li>Prepare a mixed solution of the PEG-PAsp(DET) block copolymer and the PAsp(DET) homopolymer at an equal molar ratio of residual amino groups in 10 mM HEPES buffer (pH 7.3) by adjusting the polymer concentration to 33.3 &#181;g/ml&#160;and 19.1 &#181;g/ml, respectively. &#160; 
	NOTE: The polymer concentrations described above are representative values for preparing nanomicelles with a residual molar ratio of the total amino groups in the two polymers to the phosphate groups in the pDNA (N/P ratio) of 10. The N/P ratio can vary depending on the cell type and the purpose (for details, see Discussion). The combined use of the two polymers can achieve both effective PEG shielding and functioning of PAsp(DET) to enhance endosomal escape (for details, see Discussion)18.</li>
	<li>Prepare the pDNA encoding Gaussia luciferase, GL4 luciferase, or erythropoietin by cloning the segment expressing the respective genes into the pCAG-GS plasmid (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html)&#160;to obtain expression under the CAG promoter/enhancer using a commercial kit according to manufacturer&#8217;s protocol. Amplify the pDNA in a competent Escherichia coli strain and purify it using an endotoxin-free plasmid DNA purification system. Determine the pDNA concentration at an absorbance of 260 nm to obtain a 150 &#181;g/ml&#160;solution in 10 mM HEPES buffer (pH 7.3).</li>
	<li>To prepare the polyplex nanomicelles, thoroughly mix the pDNA solution (150 &#181;g/ml&#160;in 10 mM HEPES buffer) and the premixed solution of the two polymers at a ratio of 2:1 (by volume).</li>
</ol>
4. Gene Transfection into Spheroids
<ol>
	<li>Incubate the cells (hepatocytes or MSCs) for 72 hrs after seeding onto the micropatterned plates to allow for the formation of mature spheroids. For gene transfection, add 100 &#181;l&#160;of the polyplex nanomicelle solution (containing 10 &#181;g of pDNA) to each well after replacing the culture medium with 1 ml&#160;of fresh medium. Continue the incubation with the nanomicelle solution for 24 hrs.</li>
	<li>For a control using a lipid-based transfection reagent, mix pDNA solutions with the reagent at a weight ratio reagent / pDNA&#160;of 3. Adjust the final dose of pDNA to be equal for both the lipid-based reagent and nanomicelle methods.</li>
</ol>
5. Recovery and Transplantation of Cell Spheroids
<ol>
	<li>Replace the culture medium with 200 &#181;l of chilled PBS and place the plates on ice. &#160; 
	NOTE: Generally, the spheroids can detach in about 15 min and can be recovered in the form of a suspension for transplantation.</li>
	<li>Gently aspirate the cells in the 200 &#181;l suspension using a syringe with a 23 G&#160;or 27 G needle for in vivo injections.</li>
</ol>
6. Evaluation of Transgene Expression
<ol>
	<li>For the in vitro evaluation of the expression of Gaussia luciferase secreted in the culture medium, collect 50 &#8211; 100 &#181;l&#160;of the medium precisely 24 hrs after replacing with fresh medium. Estimate the luciferase expression using a commercial renilla luciferase assay system and a luminometer according to manufacturer&#8217;s protocol. &#160; 
	NOTE: The Gaussia luciferase remains stable in the culture medium for more than a week. Thus, to evaluate the real-time efficiency of the transgene expression, replace the medium with fresh one prior to collecting the sample medium. The timing of the medium change can be flexible.</li>
	<li>For the in vivo evaluation of the transgene expression in host animals following cell transplantation, anesthetize BALB/c nude mice (female; 7 weeks old) under inhalation anesthesia with isoflurane.
	<ol>
		<li>Place a mouse in a chamber connected to an anesthesia machine to provide isoflurane for the chamber. Take out the mouse after falling asleep, and set it on the operating table with ventilation using a mask. Control the isoflurane flow approximately at 0.2&#160;&#8211; 0.5&#160;L/min by checking the conditions of the mouse.</li>
		<li>Inject 200 &#181;l&#160;of the cell suspension containing spheroids transfected with the GL4 luciferase-expressing pDNA (as described in 4.1) into the subcutaneous tissue of the abdominal region.</li>
		<li>Immediately after injecting D-luciferin (150 mg/kg; intravenous route), measure the luciferase expression using IVIS imaging system according to manufacturer&#8217;s protocol.</li>
	</ol>
	</li>
	<li>For evaluating the therapeutic effects of the cell transplantation, inject 200 &#181;l&#160;of the cell suspension containing spheroids transfected with the erythropoietin-encoding pDNA into the subcutaneous tissue of the abdominal region.
	<ol>
		<li>Collect blood samples by submandibular bleeding to obtain approximately 200 &#181;l&#160;of blood19. Measure the hemoglobin and hematocrit using a blood sample analyzer. &#160; 
		NOTE: The cell number for transplantation is regulated by the number seeded on the plates. Unfortunately, it is difficult to determine the exact cell number because the number inside spheroids cannot be measured.</li>
	</ol>
	</li>
	<li>After experiments, place the mice on a heating pad connected with a temperature controller until awakening from anesthesia.</li>
</ol>

<ol>
	<li>Landry, J., Bernier, D., Ouellet, C., Goyette, R., Marceau, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroidal+aggregate+culture+of+rat+liver+cells:+histotypic+reorganization,+biomatrix+deposition,+and+maintenance+of+functional+activities.">Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.</a> J Cell Biol. 101 (3), 914-923 (1985).</li><li>Yuasa, C., Tomita, Y., Shono, M., Ishimura, K., Ichihara, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+cell+aggregation+for+expression+of+liver+functions+and+regeneration+demonstrated+with+primary+cultured+hepatocytes.">Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.</a> J Cell Physiol. 156 (3), 522-530 (1993).</li><li>Otsuka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+multiarray+formation+of+hepatocyte+spheroids+on+a+microfabricated+PEG-brush+surface.">Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.</a> Chembiochem. 5 (6), 850-855 (2004).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+spheroid+culture+system+on+micropatterned+substrates+for+improved+differentiation+efficiency+of+multipotent+mesenchymal+stem+cells.">3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells.</a> Biomaterials. 30 (14), 2705-2715 (2009).</li><li>Bartosh, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+of+human+mesenchymal+stromal+cells+(MSCs)+into+3D+spheroids+enhances+their+antiinflammatory+properties.">Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties.</a> Proc Natl Acad Sci U S A. 107 (31), 13724-13729 (2010).</li><li>Frith, J. E., Thomson, B., Genever, P. 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B., Christensen, A. K., Gibbs, F. A., Pesch, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver.">The enzymatic preparation of isolated intact parenchymal cells from rat liver.</a> J Cell Biol. 35 (3), 675-684 (1967).</li><li>Berry, M. N., Friend, D. 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The authors declare that they have no competing financial interests.

In this protocol, it is critical to maintain the 3D structure of spheroids during the steps of gene introduction and spheroid recovery. It is essential to maintain a favorable microenvironments for the cells to avoid cell death or loss of cell activity. For example, albumin secretion, a representative innate function of hepatocytes, is well preserved in the hepatocyte spheroids, while the hepatocytes in the conventional monolayer culture rapidly lose their secretory capacity a few days after seeding12. For MSC...

Cell transplantation therapy has attracted widespread attention for treating various intractable diseases. The activity and half-life of bioactive factors that are secreted by the transplanted cells are essential for improved therapeutic effectiveness of a cell transplantation system. Genetic modification of the cells prior to transplantation is a beneficial technique to regulate and manipulate cellular functions, including the secretion of the bioactive factors. It is also important to maintain a favorable microenvironment for the cells for avoiding cell death or loss of cell activity. Three-dimensional (3D) spheroid cell culture, in which cell-to-cell interactions are well preserved, is promising for this purpose, for example, for improving albumin secretion from primary hepatocytes and promoting multi-lineage differentiation from mesenchymal stem cells (MSCs)&#160;1-7.
In this study, a novel combination system of spheroid culture and gene transfection is used to serve as a platform for genetically&#160;modified cell transplantation. For creating spheroid cells, a spheroid culture system on micropatterned culture plates is used. On these plates, cell adhesion areas of 100 &#181;m diameter are regularly arrayed in a two-dimensional manner and are surrounded by non-adhesive areas coated by a PEG matrix3. By seeding an adequate number of cells, arrays of 3D spheroids of 100 &#181;m in diameter are formed corresponding to the micropatterned culture bed.
The spheroids are recovered without disrupting their 3D structure by using thermosensitive cell-culture plates, that were coated with a thermosensitive polymer, poly(iso-propylacrylamide) (PIPAAm)&#160;8-10. The micropatterned architecture is constructed on the thermosensitive plates (custom-built). By simply lowering the temperature of the plates, the spheroids are detached from the culture bed and dispersed in phosphate buffered saline (PBS). Thus, a large number of spheroids with a uniform size of 100 &#181;m can be obtained in the form of an injectable suspension.
<img alt="Figure 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> Figure 1.&#160;Schematic representation of the spheroid culture system on a micropatterned plate.&#160;Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle. It is composed of plasmid DNA (pDNA) and polyethylene glycol (PEG)-polycation block copolymers11. These have a characteristic core-shell structure consisting of a PEG shell and an inner core of condensed pDNA, allowing safe and effective gene introduction into cells for therapeutic purposes11. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52384/52384fig2.jpg" /> 
Figure 2.&#160;Structure of the polyplex nanomicelle formed by the complex of nucleic acids and PEG-block-polycation block copolymers.&#160;In this study, the primary advantage of this technique is that the spheroid structure is not disrupted during gene transfection by the nanomicelles. After nanomicelle-mediated transfections of rat primary hepatocyte spheroids, prolonged transgene expression is obtained for more than a month with continuous albumin secretion from the hepatocytes at a level comparable to that of untransfected spheroids12. The transgene expression and albumin secretion from the spheroids are also maintained after recovery from the thermosensitive plates. It is evident that nanomicelles can safely facilitate gene introduction without impairing the innate functions of the hepatocytes. Thus, the combination of spheroid cells cultured on thermosensitive micropatterned plates with gene introduction using nanomicelles is a promising platform for genetically modified cell transplantation. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

Gene transfection of the Gaussia luciferase-expressing pDNA was performed in the spheroids formed by the hepatocytes or MSCs using polyplex nanomicelles or the control lipid-based transfection reagent12. The nanomicelles induced almost no change in the spheroid structure compared with non-transfected spheroids on the micropatterned plates, whereas the control reagent significantly disrupted the structure a day after the transfection (Figure 3). After transfection using the nanomicelles, consis...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K 次观看.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

视频: Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

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.

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

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...