The authors acknowledge funding from the National Institutes of Health R21CA182333.

1. Preparation of Polymeric Aqueous Two Phase System (ATPS)
<ol>
	<li>Weigh 0.5 g of polyethylene glycol (PEG) (MW: 35,000) and add it to 9.5 ml of complete growth medium in a sterile 15 ml conical to prepare 10 ml of 5% (w/v) aqueous PEG phase. 
	Note: Adding half of the medium to the conical first followed by adding the polymer and then the remaining amount of medium minimizes adhesion of the polymer to the conical walls and helps the polymer dissolve faster.</li>
	<li>Weigh 0.128 g of dextran (DEX) (MW: 500,000) and add it to 0.872 ml of complete growth medium in a sterile 1.5 ml microcentrifuge tube to prepare 1 ml of 12.8% (w/v) aqueous DEX phase.</li>
	<li>Vortex both solutions for about 1 min to dissolve the polymers in the medium.</li>
	<li>Keep both solutions in a water bath at 37 &#176;C for 1 hr to ensure dissolution and homogenous mixtures. Keep the caps above the water level to avoid the possibility of contamination of solutions from the bath water.</li>
	<li>Load the PEG phase solution into a sterile, plastic syringe and pass it through a syringe filter of 0.2 &#181;m pore size to remove impurities. 
	Note: Fill the syringe with the PEG solution, place it in the insert of the filter on top of a conical tube and using force, slowly push the solution through the filter. It is normal to experience resistance against movement of the syringe plunger. Minor loss of the polymer solution occurs as some of the solution will remain in the filter, but the polymer concentration will not change.</li>
	<li>Pipette 10 &#181;l of the DEX phase solution and dilute it in 10 &#181;l of medium in a separate tube to result in 6.4% (w/v) DEX phase solution. Aspirate 100 &#181;l of the PEG phase solution and dispense it into a Petri dish.
	<ol>
		<li>Dispense 0.5 &#181;l of the 6.4% (w/v) DEX phase solution into the PEG phase solution. Visually confirm successful ATPS formation by observing a DEX drop under a microscope. The drop boundary should remain visible, indicating presence of two stable, separate aqueous phases.</li>
	</ol>
	</li>
	<li>Store the stock aqueous PEG and DEX phase solutions in 4 &#176;C until use. 
	Note: It is recommended that polymer solutions are prepared fresh prior to each experiment and used within 24 hr of storage.</li>
</ol>
2. Preparation for Printing of Cancer Cell Spheroids
<ol>
	<li>Grow cancer cells of interest (e.g., MDA-MB-157 breast cancer cells) to a 90%-100% confluent monolayer. For MDA-MB-157 cells, use a medium composed of DMEM containing 10% FBS, 1% glutamine, and 1% antibiotic.
	<ol>
		<li>Harvest cells using a cell dissociation buffer (according to manufacturer&#8217;s protocol) and load the suspension into a 15 ml conical. Centrifuge it for 5 min at 173.3 x g, remove supernatant, and resuspend cells in 1 ml of complete growth medium.</li>
	</ol>
	</li>
	<li>Load cells onto a hemocytometer and count them to calculate the required number of cells for a desired spheroid cell density. A confluent monolayer of MDA-MB-157 cells grown in a T75 flask usually gives ~7 x 106 cells. 
	Note: Required cell density for a drop volume to generate a single spheroid will depend on the cell type.22 For example, a density of 1.5 x 104 or larger for MDA-MB-157 cells is recommended per 0.3 &#181;l DEX phase drop to ensure formation of a single spheroid.</li>
	<li>Centrifuge cells for a second time for 5 min at 173.3 x g and resuspend them in an appropriate volume of growth medium to concentrate the suspension to a desired cell density. 
	Note: For example if 7 x 106 cancer cells were harvested, the total volume of cell suspension required to form a spheroid of 1.5 x 104 cell density in a 0.3 &#181;l DEX phase drop will be 140 &#181;l. However due to dilution with the DEX phase solution in the next step, only use 70 &#181;l of cell culture medium to resuspend cells.</li>
	<li>Add to the resulting cell suspension an equal volume of the 12.8% (w/v) aqueous DEX phase solution prepared in 1.2. For the example of 1.5 x 104 cell density spheroid, 70 &#181;l of the DEX phase solution is added to 70 &#181;l of cell suspension.</li>
	<li>Thoroughly mix the cell suspension to ensure uniform distribution of cells and mixing of DEX solution. Pipetting up and down should be done gently to prevent bubble formation.</li>
</ol>
3. Preparation for Printing of Co-cultured Spheroids
<ol>
	<li>Grow cancer cells (e.g., MDA-MB-157 human breast cancer cells) and support cells (e.g., human fibroblasts) to a 90%-100% confluent monolayer. Harvest each cell type using a cell dissociation buffer (according to manufacturer&#8217;s protocol).
	<ol>
		<li>Load each suspension into a 15 ml conical, centrifuge them for 5 min at 173.3 x g, and aspirate supernatant from each conical. Resuspend cells of each conical in 1 ml of complete growth medium. 
		Note: Fluorescent dyes such as Calcein AM (live cell stain) and nuclear dyes (Hoechst) can be used to distinguish the two cell types.</li>
	</ol>
	</li>
	<li>Load each cell type separately onto a hemocytometer and count them to calculate the required number of each cell type for a desired ratio of cancer cells to supporting cells in co-cultured spheroids. Confluent monolayers of MDA-MB-157 cells and fibroblasts grown in T75 flasks usually give ~7 x 106 and ~6 x 106 cells, respectively.</li>
	<li>Add the correct volume from the suspension of supporting cells to cancer cells suspension to give a desired ratio of the number of two cell types. For example, use a ratio of 50 cancer cells to 1 fibroblast cell.</li>
	<li>Centrifuge the conical containing the mixed cell suspension for 5 min at 173.3 x g and resuspend cells in an appropriate volume to result in final density comprising of equal volumes of growth medium and the 12.8% (w/v) aqueous DEX phase solution (prepared in 1.2).</li>
	<li>Gently pipette the resulting cell suspension up and down to ensure homogeneity of the suspension.</li>
</ol>
4. Printing of Tumor Spheroids into a 96-well Plate
<ol>
	<li>One day prior to experiments, coat non-treated, round-bottom 96-well plates with 1% (w/v) Pluronic at 37 &#176;C for 24 hr. This coating prevents cell attachment over the culture period.</li>
	<li>Dispense 50 &#181;l of the filtered 5% (w/v) aqueous PEG phase into each well of a 96-well plate (destination plate).</li>
	<li>Using a pipette, mix the cell suspension (prepared in step 2 or step 3 for mono- and co-culture, respectively) and add 20 &#181;l of the suspension to every other well from one column of a 384-well plate (source plate).</li>
	<li>Turn on the liquid handler and &#8220;home&#8221; the pipetting head to register coordinates. Then compile a previously defined protocol (below in 4.6) to ensure labware and parameters are defined correctly. This &#8220;homing&#8221; step may be different for different liquid handlers. 
	Note: The flow of the protocol, shown step-by-step below in 4.6, will be similar regardless of the robotic liquid handler used; however, the programming interface may look different due to the use of different software.</li>
	<li>Place the source plate, the destination plate, and pipette tip boxes at defined positions on the workstation of the liquid handler as shown in Figure 1.</li>
	<li>Execute the protocol that includes the following steps:
	<ol>
		<li>Load one column of barrels from the pipetting head of the liquid handler with mixing pipette tips (8 tips) from the workstation (Figure 1).</li>
		<li>Mix the cell suspension in wells of the source plate (Figure 1). Select a mixing volume smaller than the cell suspension volume in wells to avoid bubble formation.</li>
		<li>Eject tips into an empty waste tips box (Figure 1).</li>
		<li>Load one column of barrels from the pipetting head with 10 &#181;l dispensing pipette tips (Figure 1).</li>
		<li>Aspirate 0.3 &#181;l of the cell suspension from the source plate (Figure 1) into each pipette tip.</li>
		<li>Dispense the cell suspension into wells of one column of the destination plate (Figure 1). Use a dispense height of 0.5 mm and a dispense flow rate of smaller than 1 &#181;l/sec.</li>
		<li>Repeat steps 4.6.1 through 4.6.6 until the column-by-column printing into the entire destination plate is complete.</li>
	</ol>
	</li>
	<li>Carefully remove the destination plate from the workstation surface and place it in a humid incubator at 37 &#176;C and 5% CO2. Maintain the plate horizontal when carrying it into the incubator to avoid disrupting of DEX phase drops containing cells.</li>
	<li>Incubate the plate for 24 hr to allow aggregation of cells into a spheroid within the DEX phase drop.</li>
</ol>
5. Drug Treatment of Cancer Cell Spheroids
Note that the following protocol is for a 4-day drug treatment and includes a renewal with fresh drug after day 2. It can be modified for other treatment periods.
<ol>
	<li>Visually confirm spheroid formation in 96-well plates after 24 hr. If needed, image wells for measurement of size of spheroids by averaging the smallest and largest diameters of each spheroid.</li>
	<li>Prepare the stock solution of a desired drug by dissolving it in a solvent recommended by the manufacturer at a predetermined solubility concentration. For example, dissolve cisplatin in water at 2 mg/ml. 
	Note: Protect the stock solution of the drug from light if the drug is light-sensitive.</li>
	<li>Using the serial dilution technique, prepare diluted drug concentrations with cell culture medium from the stock solution prepared in the previous step. Each dilution should be made twice the desired concentration. Dilution ratios will vary depending on the starting stock concentration and desired working concentrations.</li>
	<li>Add 50 &#181;l of drug solution prepared in the previous step to each well. This can be done robotically or by manual pipetting. 
	Note: Each drug concentration will be diluted in half to the desired concentration by the 50 &#181;l aqueous PEG phase already in each well.
	<ol>
		<li>Use spheroids from two columns of a 96-well plate (n=16) for each concentration.</li>
		<li>In control wells, add only 50 &#181;l of fresh culture medium to the existing 50 &#181;l of the aqueous PEG phase.</li>
	</ol>
	</li>
	<li>Incubate spheroids with the drug for 48 hr at 37 &#176;C and 5% CO2, protected from light.</li>
	<li>After 48 hr of incubation, prepare fresh dilutions of the drug at desired concentrations and renew drug by adding 50 &#181;l of fresh drug solution to each well. 
	Note: The wells already contain the desired drug concentration from the first drug treatment. Therefore in this renewal phase, each concentration is prepared at the desired concentration since no dilution will occur.</li>
	<li>Incubate spheroids with the drug for another 48 hr at 37 &#176;C and 5% CO2, protected from light.</li>
</ol>
6. Analysis of Cellular Viability in Spheroids
<ol>
	<li>After 48 hr of incubation with renewed drug (i.e., total time of treatment of spheroids with a drug is 4 days), measure the total volume of medium in one well by manual pipetting. 
	Note: Typical volume in one well is ~140 &#181;l (a small decrease occurs due to evaporation).</li>
	<li>Calculate the volume of cell viability reagent (e.g., PrestoBlue) as 10% concentration of the total well volume. Add this volume of cell viability reagent to each well. 
	Note: With an existing media volume of 140 &#181;l in a well, a volume of 15.6 &#181;l is required to give a 10% concentration of the total well. This is calculated by dividing the existing media volume by 9 (140 &#181;l / 9 = 15.6 &#181;l).</li>
	<li>Incubate the plate with the cell viability reagent added to each well for 6 hr at 37 &#176;C, protected from light.</li>
	<li>Place the plate in a micro-plate reader and read the fluorescence signal at 560 nm and 590 nm excitation and emission wavelengths, respectively.</li>
	<li>Calculate the percent viability of cells in spheroids by normalizing the fluorescent signal from treated wells to that of control wells after averaging the values from the same treatment conditions.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+fibroblasts+stimulate+invasion+of+breast+cancer+cells+in+a+three-dimensional+culture+and+increase+stroma+development+in+mouse+xenografts.">Human mammary fibroblasts stimulate invasion of breast cancer cells in a three-dimensional culture and increase stroma development in mouse xenografts.</a> BMC Cancer. 10, 444 (2010).</li><li>Ozdemir, B. C., 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=Depletion+of+carcinoma-associated+fibroblasts+and+fibrosis+induces+immunosuppression+and+accelerates+pancreas+cancer+with+reduced+survival.">Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.</a> Cancer Cell. 25, 719-734 (2014).</li><li>Hsiao, A. Y., 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=Microfluidic+system+for+formation+of+PC-3+prostate+cancer+co-culture+spheroids.">Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids.</a> Biomaterials. 30, 3020-3027 (2009).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, 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=Spheroid-based+drug+screen:+considerations+and+practical+approach.">Spheroid-based drug screen: considerations and practical approach.</a> Nat. Protoc. 4, 309-324 (2009).</li><li>Weigelt, B., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+microenvironmental+influences+on+the+normal+mammary+gland+and+breast+cancer.">Unraveling the microenvironmental influences on the normal mammary gland and breast cancer.</a> Semin. Cancer Biol. 18, 311-321 (2008).</li><li>Mueller, M. M., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friends+or+foes+-+bipolar+effects+of+the+tumour+stroma+in+cancer.">Friends or foes - bipolar effects of the tumour stroma in cancer.</a> Nature Reviews Cance. 4, 839-849 (2004).</li><li>Bissell, M. J., Radisky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putting+tumours+in+context.">Putting tumours in context.</a> Nat. Rev. Cancer. 1, 46-54 (2001).</li></ol>

The authors have nothing to disclose.

Spheroids present a realistic model to better understand tumor physiology and drug efficacy and provide a useful tool for anti-cancer drug discovery. Such applications would greatly benefit from simple spheroid generation and maintenance techniques that only require standard labware, liquid handling tools and screening equipment. The use of an aqueous two-phase system to spontaneously aggregate cancer cells within the drop phase allows efficient production and maintenance of spheroids with robotic liquid handlers, and <e...

Cell-based assays provide an important tool for the development and discovery of new anti-cancer drugs.1,2 Historically, monolayer cultures of cancer cells have been employed to investigate the efficacy of candidate compounds against particular types of cancer cells. The ease of maintenance of monolayer cultures in standard culture plates, the compatibility of standard plates with commercial robotic tools for addition of reagents, and with screening equipment for downstream analysis of cellular responses to chemical compounds are the major benefits that render 2D cultures an attractive tool for drug testing.3 Unfortunately, monolayer cell assays often fail to predict the efficacy of compounds in vivo, making drug development and discovery an extremely costly process.4,5 Despite significant investment and effort by pharmaceutical companies and academic units, only ~1% of anti-cancer drugs in clinical trials were approved by the FDA over the past two decades.6 Disparity between 2D cultures and the complex 3D environment of cancer cells in vivo is a major shortcoming of monolayer culture systems.7 Therefore, screening of candidate compounds against tumor cells in a setting that more closely resembles the 3D tumor environment may expedite development of novel chemotherapy drugs.8
Cancer cell spheroids present a relevant 3D tumor model in vitro.9,10 Spheroids are compact clusters that form through spontaneous or induced assembly of cancer cells on non-adherent surfaces or in suspension using techniques such as spinner flask, liquid overlay, microfabricated micro-well arrays, microfluidics, and hanging drops.11-16 Spheroids mimic key features of solid tumors including geometry and limited transport of oxygen, nutrients, and drug compounds into the central zone; hence, they more closely regenerate drug response of solid tumors compared to monolayer cultures.17-19 Despite this marked benefit, spheroids are not routinely used for screening of chemical compounds against cancer cells. Difficulty of producing uniform sized spheroids in a standard high throughput setting that is compatible with commercially available robotics and screening/imaging tools impedes incorporation of spheroid culture into drug development pipeline. Although custom materials and plates have recently become commercially available to address this need, cost considerations deter their widespread use.
Two major techniques with the capability of producing consistent sized spheroids in high throughput use a new hanging drop platform and microfabricated micro-wells.13,16,20 However, both approaches require special plates and devices that are expensive to fabricate and inconvenient for endpoint users in core research centers and pharmaceutical industries where the most major efforts for the discovery of new anti-cancer drugs are made. Despite some improvements in the stability of cell-containing drops with a recent design of hanging drop plates, only every other hole of the plate is still used during culture to avoid spreading/merging of drops.16 This significantly decreases experimental throughput. Drug addition and renewal is difficult with manual or robotic pipetting and spheroids need to be transferred into a standard plate for biochemical analysis because this plate configuration is not readily compatible with conventional screening equipment such as plate readers.21 Micro-wells fabricated using soft lithography also allow controlled size spheroid production.13,20 However, incompatibility of this platform with standard pipetting tools prevents treating of individual spheroids with different drug compounds/concentrations, exposing all spheroids to a single treatment condition. Thus, this method is not appropriate for high throughput compound screening that requires simultaneous testing of multiple compounds/concentrations.
To overcome these obstacles, a new technique for high throughput production of consistently sized cancer cell spheroids in standard 96-well plates has been developed.22,23 The approach is based on a polymeric aqueous two-phase system (ATPS) with polyethylene glycol (PEG) and dextran (DEX) as phase-forming polymers.24 ATPSs have recently been utilized in a variety of novel cell biological applications to enable cell micropatterning and localized delivery of biological reagents to cells in highly aqueous media.25-32 To form a spheroid, cancer cells are mixed with the aqueous DEX phase and a sub-microliter drop of the resulting suspension is pipetted into a well containing the immersion aqueous PEG phase solution. The drop remains immiscible from the immersion phase and confines cells to facilitate formation of a spheroid. Importantly, the highly aqueous immersion phase provides nutrients to cells of the spheroid and minimizes the well-known problem of media evaporation common to some other assays that causes changes in media osmolality and fluctuations of drug concentrations. This technique enables spheroid production and drug treatment only using commercially-available reagents and pipetting tools in standard 96-well plates. Importantly, analysis of cellular responses of spheroids is performed in the same plate using standard biochemical assays and plate readers. The ease of working with ATPS and adaptability of the approach to robotic liquid handling makes high throughput generation of both mono-culture and co-culture spheroids a straightforward laboratory technique. This new approach will be a major step forward toward integration of cancer cell spheroids into drug development and discovery processes with improved testing throughput and cost-effectiveness (increasing numbers of tested compounds and reduced reagent consumption) and efficiency (reducing hands-on time).
A detailed protocol for robotic production of cancer cell spheroids in 96-well plates using the ATPS approach is described below. In addition, details of drug treatment of resulting spheroids and downstream analysis of cellular responses using a commercial biochemical assay are presented.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents and Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol, Mw: 35,000</td>
			<td>Sigma-Aldrich</td>
			<td>94646</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Mw: 500,000</td>
			<td>Pharmacosmos</td>
			<td>5510 0500 9007</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12306C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Clacein AM</td>
			<td>Life Technologies</td>
			<td>C3100MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Life Technologies</td>
			<td>33342</td>
			<td></td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Spectrum Chemicals</td>
			<td>15663-27-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PrestoBlue</td>
			<td>Life Technologies</td>
			<td>A-13261</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-108</td>
			<td>Sigma Aldrich</td>
			<td>542342</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Tips (10 &#181;l)</td>
			<td>Fluotics</td>
			<td>C-P10V11.ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Tips (70 &#181;l)</td>
			<td>Fluotics</td>
			<td>C-P70V11.ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom 96-well plates</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Handler</td>
			<td>Agilent Technologies</td>
			<td>SRT Bravo</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Biotek Instruments</td>
			<td>Synergy H1M</td>
			<td></td>
		</tr>
	</tbody>
</table>

robotic production of cancer cell spheroids with an aqueous two-phase system for drug testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

The workstation of the robotic liquid handler is shown in Figure 1. The pipetting head and all stations used in the robotic printing of spheroids in section 4.6 are labeled. The image shows the use of two different stations for tip boxes (one set of tips for mixing and the second set for aspirating/dispensing of cell suspension&#8211;aqueous DEX phase mixture). The entire setup is housed within a standard biological safety cabinet to maintain sterility. Figure 2 depicts a schematic of th...

Watch this Scientific Journal Video about Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing at JoVE.com

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

robotic-production-cancer-cell-spheroids-with-an-aqueous-two-phase

Research

JoVE Journal

Bioengineering

8.9K Views.  The University of Akron. A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Video: Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

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

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

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

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

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Scalable Generation of Uniform Cell Spheroids Using a 3D Acoustic Assembly Device

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

Author Spotlight: Development of a Scaffold-Free Acoustic Assembly Method for High-Quality 3D Cell Spheroid Culture 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...

Setting Up an Acoustic Assembly System and Cell Culture Harvesting for Generating Uniform Cell Spheroids

To begin, sterilize the chamber of the acoustic device with 75%alcohol for five minutes. Then, clean the device with sterile PBS and irradiate it with UV light for one hour. Mount the sterile acoustic device onto a microscope stage for top-view observation of the chamber's interior.Position a digital microscope on the side of the device that is free from PZT transducers, enabling side-view observation of the chamber's interior. Independently connect the wires from the three PZT transducers in series to three power amplifiers and three output channels of function generators. Program the settings on the function generators for each PCT transducer, specifying parameters such as sinusoidal waveforms, frequency, and amplitude.Culture 3CA cells in DMEM supplemented with 10%FBS and 1%penicillin streptomycin in a T25 cell culture flask. When the cells become 80%confluent, wash the culture twice with PBS. After removing the PBS, add two milliliters of 0.05%trypsin-EDTA to the flask and incubate at 37 degrees Celsius for cell detachment.Add two milliliters of complete cell culture medium into the flask to stop the trypsinization. Transfer the cell suspension to a 15-milliliter tube and centrifuge at 200 G for five minutes.