Name: gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation
Uploaded: 2015-07-31T13:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+three-dimensional+culture+methods+enhance+mesenchymal+stem+cell+properties+and+increase+therapeutic+potential.">Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential.</a> Tissue Eng Part C Methods. 16 (4), 735-749 (2010).</li><li>Nakasone, Y., Yamamoto, M., Tateishi, T., Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+spheroids+underlayered+with+nonparenchymal+cells+for+biomedical+applications.">Hepatocyte spheroids underlayered with nonparenchymal cells for biomedical applications.</a> IEICE Transactions on Electronics. E94, 176-180 (2011).</li><li>Nishida, K., 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=Corneal+reconstruction+with+tissue-engineered+cell+sheets+composed+of+autologous+oral+mucosal+epithelium.">Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium.</a> N Engl J Med. 351 (12), 1187-1196 (2004).</li><li>Ohashi, K., 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=Engineering+functional+two-+and+three-dimensional+liver+systems+in+vivo+using+hepatic+tissue+sheets.">Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets.</a> Nat Med. 13 (7), 880-885 (2007).</li><li>Sekine, 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=Cardiac+cell+sheet+transplantation+improves+damaged+heart+function+via+superior+cell+survival+in+comparison+with+dissociated+cell+injection.">Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection.</a> Tissue Eng Part A. 17 (23-24), 2973-2980 (2011).</li><li>Itaka, K., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+prospects+of+polyplex+nanomicelles+for+plasmid+DNA+delivery.">Progress and prospects of polyplex nanomicelles for plasmid DNA delivery.</a> Curr Gene Ther. 11 (6), 457-465 (2011).</li><li>Endo, T., Itaka, K., Shioyama, M., Uchida, S., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfection+to+spheroid+culture+system+on+micropatterned+culture+plate+by+polyplex+nanomicelle:+a+novel+platform+of+genetically-modified+cell+transplantation.">Gene transfection to spheroid culture system on micropatterned culture plate by polyplex nanomicelle: a novel platform of genetically-modified cell transplantation.</a> Drug Deliv and Transl Res. 2 (5), 398-405 (2012).</li><li>Howard, R. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J Cell Biol. 43 (3), 506-520 (1969).</li><li>Tateno, C., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cultivation+of+adult+rat+hepatocytes+that+undergo+multiple+cell+divisions+and+express+normal+parenchymal+phenotypes.">Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes.</a> Am J Pathol. 148 (2), 383-392 (1996).</li><li>Kanayama, N., 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=A+PEG-based+biocompatible+block+catiomer+with+high+buffering+capacity+for+the+construction+of+polyplex+micelles+showing+efficient+gene+transfer+toward+primary+cells.">A PEG-based biocompatible block catiomer with high buffering capacity for the construction of polyplex micelles showing efficient gene transfer toward primary cells.</a> ChemMedChem. 1 (4), 439-444 (2006).</li><li>Itaka, K., Ishii, T., Hasegawa, Y., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polyamino+acid-based+polycations+as+safe+and+effective+gene+carrier+minimizing+cumulative+toxicity.">Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity.</a> Biomaterials. 31 (13), 3707-3714 (2010).</li><li>Uchida, S., 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=PEGylated+Polyplex+With+Optimized+PEG+Shielding+Enhances+Gene+Introduction+in+Lungs+by+Minimizing+Inflammatory+Responses.">PEGylated Polyplex With Optimized PEG Shielding Enhances Gene Introduction in Lungs by Minimizing Inflammatory Responses.</a> Mol Ther. 20 (6), 1196-1203 (2012).</li><li>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+simple,+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet.">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Anim (NY). 34, 39-43 (2005).</li><li>Uchida, S., 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=An+injectable+spheroid+system+with+genetic+modification+for+cell+transplantation+therapy.">An injectable spheroid system with genetic modification for cell transplantation therapy.</a> Biomaterials. 35 (8), 2499-2506 (2014).</li></ol>

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 MSCs, the spheroids significantly enhance their efficiency of differentiation into adipocytes or osteoblasts, with increased expression levels of genes involved in adipogenesis and osteogenesis, and down-regulation of genes that maintain the MSC self-renewal phenotype4.
For gene introduction, various transfection reagents had been tested previously, including lipid-based ones (Figure 3, 4) and polymer-based ones, such as polyethylenimine. Unfortunately, none of the reagents provided successful gene introduction without disrupting the spheroid structure. Especially, the lipid-based reagents tend to induce complete disruption of the spheroids, although the transgene expression is efficient, even in the remaining cells on the micropatterned plates. It is likely that the lipid-based reagents induce significant membrane destabilization, leading to high transgene expression, but disrupt the spheroids by rendering the cell-to-cell interactions unstable.
In contrast, the cationic polymer disrupted the spheroids to a lesser extent than the lipids, especially in the conditions of low N/P ratios, to form the polyplexes. The standard protocol shown here uses polyplex nanomicelles that possess PEG shielding. Alternatively, polyplexes with cationic homopolymers, such as polyethylenimine, can also be used for gene introduction into the spheroids. Indeed, cationic polyplexes without the PEG surface generally provide high transgene expression in in vitro transfections, and are attractive options depending on the cell type and purpose of the spheroid transplantation.
A characteristic feature of this protocol is the use of micropatterned culture plates coated with a thermosensitive polymer. The micropatterned arrays can produce a large number of cell spheroids with a uniform diameter. This protocol uses 100 &#181;m diameter arrays, but this aspect is flexible; however, very large diameters, such as 500 &#181;m, often cause cell necrosis in the core of the spheroids (unpublished data). By simply cooling the plates on ice, the spheroids can be obtained in the form of an injectable suspension by gentle aspiration with a syringe. A potential limitation of this technique is the size of the needle used to inject the spheroids. It was confirmed that a 27-G needle can be safely used for spheroids with 100 &#181;m diameter. However, narrow needles cannot be applied for these spheroids. Since a 27-G needle is too large, especially for transplanting spheroids into mouse spinal cords, a possible alternative is to use scaffolds to retain cells inside them.
This protocol can be potentially applied for several purposes of cell transplantation. Compared to the suspension spheroid culture system, the micropatterned plates have the advantage that a sufficient number of spheroids with uniform diameter can be easily obtained. Although the method of gene introduction can be flexible, as mentioned previously, polyplex nanomicelles with PEG shielding are particularly effective for avoiding spheroid disruption. Hopefully, this system will improve the therapeutic effects of transplanted cells.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><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&amp;rsquo;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&lt;sup&gt;)&lt;/sup&gt;</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>&lt;em&gt;Escherichia coli&lt;/em&gt; DH5&amp;alpha; 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&amp;trade; 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, consistent albumin secretion and the transgene (Gaussia luciferase) expression were well maintained for almost a month. However, when the control reagent was used, no albumin secretion was observed, although the degree of the transgene expression was similar to that obtained from the nanomicelles (Figure 4)&#160;12.
During spheroid recovery using the thermosensitive micropatterned plates, the 3D structure of the spheroids was well preserved for both the primary hepatocytes and the MSCs in suspension after recovery from the cooled plates (Figure 5). The structure was also well maintained after passing the spheroids through injection needles with a sufficiently large caliber, such as the 23 (400 &#181;m) and 27&#160;G (220 &#181;m) needles.
After subcutaneous injections of the recovered hepatocyte spheroids, luciferase expression was detected in the animals for more than a few weeks (Figure 6)&#160;20. Albumin expression from transplanted hepatocytes was observed in the host tissue20, suggesting that the cell function was preserved through the process of spheroid recovery and transplantation. To test their potential for therapeutic applications, the hepatocyte spheroids were also transfected with erythropoietin-encoding pDNA. After subcutaneous transplantation of the spheroids expressing erythropoietin, a significant hematopoietic effect was obtained in the animals for one month (Figure 7)&#160;20.
<img alt="Figure 3" src="/files/ftp_upload/52384/52384fig3.jpg" /> 
Figure 3.&#160;Microscopic images of hepatocyte spheroids (A, B) and MSC spheroids (C, D) on a micropatterned plate a day after gene transfection, using polyplex nanomicelles (A, C) or lipid-based reagent (B, D). Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52384/52384fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52384/52384fig4.jpg" /> 
Figure 4.&#160;(A) Transgene expression of Gaussia luciferase after transfecting the hepatocyte spheroids. (B) Albumin secretion from the hepatocyte spheroids or hepatocytes in a monolayer culture after the transfection. Spheroids were transfected using nanomicelle (&#9679;) or control lipid-based transfection reagent (&#9675;), or naked pDNA (&#9650;). The albumin secretion from the control (untransfected) hepatocytes (in spheroids (&#9651;) or monolayer culture (&#215;)) is also shown. The results are represented as mean &#177; SEM; n = 4 for monolayer culture and n = 6 for spheroids. (Reprinted with permission from reference 12). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52384/52384fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52384/52384fig5.jpg" /> 
Figure 5.&#160;Microscopic images of the hepatocyte spheroids in suspension after passing them through (A) 23- or (B) 27&#160;G injection needles. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52384/52384fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52384/52384fig6.jpg" /> 
Figure 6.&#160;Luciferase expression in host mice after hepatocyte transplantation. After 24 hrs after transfection with luciferase (GL4)-encoding pDNA, the hepatocyte spheroids and single-cell suspension from the monolayer cultures were transplanted into the subcutaneous tissue of the abdominal region. The luciferase expression in the host mice was evaluated using an IVIS Imaging System (Reprinted with permission from reference 20).<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52384/52384fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52384/52384fig7.jpg" /> 
Figure 7.&#160;Hematopoiesis after transplantation of the erythropoietin-expressing hepatocytes. Hepatocytes in the spheroid and monolayer cultures were transfected with erythropoietin-encoding pDNA. After 24 hrs of transfection, the spheroids and single-cell suspensions from the monolayer cultures were subcutaneously transplanted into the mouse abdomen. At 22 and 28 days after transplantation, (A) hemoglobin and (B) hematocrit levels were measured from the blood samples. Data are presented as the mean &#177; SEM; n = 12 for spheroid and monolayer groups and n = 6 for untreated controls. Statistical significance was determined by a 2-tailed Student&#39;s t-test (Reprinted with permission from reference 20). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52384/52384fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.7K Views.  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.

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

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Cancer Research

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Developmental Biology

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Biology

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

Magnetically Bioprinted 3D Spheroid Co-cultures: A Method for Co-culturing Cancerous Cells and Fibroblasts

Source: Noel P. et al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/56081/" target="_blank">Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts</a>.&#160;J. Vis. Exp. (2017)
This video explains the 3D magnetic bioprinting of co-cultured pancreatic cancer cells and fibroblasts. Pancreatic cancer cells and activated pancreatic fibroblast cells are co-cultured and magnetized using a biocompatible nanoparticle assembly, to generate spheroids, under the influence of magnetic forces.

Source: Noel P. et al.&#160;Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts.&#160;J. Vis. ...

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

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