This work has been supported in part by NIH grants, R44 GM087784 and R01 HL109505. Authors thank the technical and OEM team at Gilson, INC for extended technical support.

The robotic liquid handling system, which for the following protocols was Gilson&#8217;s pipetmaX, facilitates automated, scalable stem cell culture and production while maintaining traditional adherent culture conditions in the 96-well plate format. Like traditional 6-well plate culture, iPSCs are grown with this protocol as a monolayer of distinct colonies but in the 96-well plate format. Each well can be isogenic or distinct and either configuration can be cultured in parallel since the robot can keep each well segregated. The typical robotic iPSC culture routine has two phases: a feeding program where cultures are fed on a customizable schedule and a passage program where the user chooses the dissociation method and splitting ratio thereby controlling seeding density and scale rate. The following protocols describe how a new culture is started, how feeding and passage are accomplished and the supplementary programs that facilitate iPSC culture.
Note: Please see the Materials List for specific recommendations on the matrix coating material and dissociation reagents validated for the following protocols.
1. Preparing Extracellular Matrix Gel Coated 96-well Plates
<ol>
	<li>Remove the packaging from six new, RT 96-well plates and place them on the liquid handling robot bed in positions 2, 3, 5, 7, 8, 9 (see Figure 1A for bed positions).</li>
	<li>Place a pre-chilled, 4 &#176;C 4-well trough in bed position 6.</li>
	<li>Load column 12 of the tip rack in bed position 1 with pre-chilled, 4 &#176;C 200 &#181;l pipet tips.</li>
	<li>Fill the first (left most) trough well in bed position 6 with 25 ml 4 &#176;C DMEM/F12 by pouring an aliquot using sterile technique. Alternatively, use a pipet to complete the transfer.</li>
	<li>Remove the 96-well plate lids and slide each into the specially designed plate lid holding rack (PLHR, see Figure 1A). Avoid contact with the inside of plate lids. 
	Note: Handling the inside of a plate lid or stacking the plate lids where the inside of one lid touches the outside of another is an excellent route for contamination.</li>
	<li>Using the robotic control touch pad, press the following series of buttons to start the well pre-wetting process:
	<ol>
		<li>Tap &#8220;Run a Protocol&#8221;.</li>
		<li>Select &#8220;extracellular matrix gel Prewet&#8221; and tap next.</li>
		<li>User decision: Tap &#8220;test run&#8221; to use the step-by-step wizard to pre-test the selected program. 
		Note: The robot does not run but rather the software tests the protocol for software problems. With established protocols, tapping, &#8220;skip setup&#8221; is acceptable.</li>
		<li>Tap &#8220;Run Protocol&#8221;.</li>
	</ol>
	</li>
	<li>During the pre-wetting process (step 1.6) continue to step 1.8.</li>
	<li>Thaw on ice one 4 mg aliquot of growth factor reduced extracellular matrix gel (GFRM) contained in a 50 ml conical tube stored at -80 &#176;C. Keep the aliquot on ice until ready to use. 
	Note: The GFRM will solidify if allowed to come to RT and will not be useful.</li>
	<li>Resuspend the 4 mg GFRM aliquot in 24 ml 4 &#176;C DMEM/F12 with a 25 ml glass pipet. When transferring the DMEM/F12, pause for 2-3 sec to allow the pipet to cool prior to resuspending the GFRM aliquot.</li>
	<li>When the pre-wetting process (step 1.6) is complete, continue to step 1.11.</li>
	<li>Transfer the 24 ml of DMEM/F12 GFRM mixture to the fourth well of the trough in bed position 6.</li>
	<li>Using the robotic control touch pad, press the following series of buttons to start the extracellular matrix gel coating process:
	<ol>
		<li>Tap &#8220;Run a Protocol&#8221;.</li>
		<li>Select &#8220;extracellular matrix gel aliquot&#8221; and tap next.</li>
		<li>User decision: Tap &#8220;test run&#8221; to use the step-by-step wizard to pre-test the selected program. 
		Note: The robot does not run but rather the software tests the protocol for software problems. With established protocols, tapping, &#8220;skip setup&#8221; is acceptable.</li>
		<li>Tap &#8220;Run Protocol&#8221;.</li>
	</ol>
	</li>
	<li>When step 1.12 is complete, replace the plate lids.</li>
	<li>Transfer the extracellular matrix gel coated 96-well plates to a 37 &#176;C, 5% CO2, humidified incubator and hold there for 24 hr at which point the plates will be ready to use or stored. 
	Note: Under extenuating circumstances, the newly coated extracellular matrix gel plates may be used after 15-30 min of incubation but O/N to 24 hr incubation is recommended.</li>
	<li>Repeat protocol steps 1.1, 1.3, 1.5-1.6, 1.10, and 1.12-14 until all of the DMEM/F12 with GFRM is used.</li>
</ol>
2. Storing Extracellular Matrix Gel Coated 96-well Plates
<ol>
	<li>Remove extracellular matrix gel coated 96-well plates from the 37 &#176;C, 5% CO2, humidified incubator.</li>
	<li>Optional: Allow the extracellular matrix gel coated 96-well plates to come to RT before proceeding to step 2.3. 
	Note: This optional step will reduce condensation build up when the plates are placed at 4 &#176;C.</li>
	<li>Place the plates on the liquid handling robot bed in positions 2, 3, 5, 7, 8, 9 (see Figure 1A for bed positions).</li>
	<li>Load column 12 of the tip rack in bed position 1 with RT&#160;200 &#181;l pipet tips.</li>
	<li>Place a four trough reservoir in bed position 6.</li>
	<li>Fill well 4 (rightmost) of the reservoir with RT DMEM/F12.</li>
	<li>Remove the 96-well plate lids and slide each into the PLHR.</li>
	<li>Using the robotic control touch pad, press the following combination of buttons:
	<ol>
		<li>Tap &#8220;Run a Protocol&#8221;.</li>
		<li>Select &#8220;extracellular matrix gel aliquot&#8221; and tap next.</li>
		<li>User decision: Tap &#8220;test run&#8221; to use the step-by-step wizard to pre-test the selected program. 
		Note: The robot does not run but rather the software tests the protocol for software problems. With established protocols, tapping, &#8220;skip setup&#8221; is acceptable.</li>
		<li>Tap &#8220;Run Protocol&#8221;.</li>
	</ol>
	</li>
	<li>Once complete, replace the plate lids and remove the plates from the machine bed.</li>
	<li>Wrap around the entire side of each extracellular matrix gel coated 96-well plate (where the lid meets the plate) with a one inch by 6 inch paraffin film strip. Be sure to stretch the paraffin film to create an airtight seal.</li>
	<li>Optional: Label the plates with the date of completed extracellular matrix gel coating, the extracellular matrix gel lot number, user name and any other information.</li>
	<li>Store the plates at 4 &#176;C where they will be good to use for up to two weeks. 
	Note: Plates have been successfully used beyond the two week limit, however, it is not recommended.</li>
</ol>
3. Performing a Stem Cell Colony Seeding Density Gradient in the 96-well Plate Format from a Live or Frozen Culture: How to Start or Restore Normal Passage Intervals to a Stem Cell Line
<ol>
	<li>If starting with a frozen culture, proceed to step 3.2. If starting with a live stem cell culture already on a plate, begin at step 3.5.</li>
	<li>Thaw the frozen culture by swirling the tube in a 37 &#176;C water bath until only a small chunk of ice remains.</li>
	<li>Optional: Dilute the thawed cells into 10 ml RT stem cell growth media (SCGM), centrifuge for 2 min at 300 x g, aspirate the supernatant media and resuspend the stem cell pellet in 7 ml fresh SCGM containing 10 &#181;M Y27632. 
	Note: This step eliminates carryover of the freezing media.</li>
	<li>Proceed to Step 3.6.</li>
	<li>If starting with live, already plated stem cells: passage the cells as normal using enzymatic or non-enzymatic dissociation. Try to harvest a total of 1.5-3 million cells; usually one well of a six-well plate. Centrifuge the harvested cells for 2 min at 300 x g, aspirate the supernatant media and resuspend the stem cell pellet in 7 ml fresh SCGM containing 10 &#181;M Y27632. Proceed to Step 3.6.</li>
	<li>Ensure the cells, either from a frozen or live culture, are suspended in 7 ml RT SCGM with 10 &#956;M Y27632.</li>
	<li>Load column 12 of the tip rack in bed position one with RT 200 &#181;l pipet tips.</li>
	<li>Place a four trough reservoir in bed position 2.</li>
	<li>Place one new extracellular matrix gel coated 96-well plate pre-warmed to 37 &#176;C in bed position 5 and remove the lid placing it in the PLHR.</li>
	<li>Transfer the 7 ml of resuspended cells to well 3 of the reservoir by pipet or sterile pour.
	<ol>
		<li>Using the robotic control touch pad, press the following combination of buttons:</li>
		<li>Tap &#8220;Run a Protocol&#8221;.</li>
		<li>Select &#8220;Seeding Density Gradient&#8221; and tap next.</li>
		<li>User decision: Tap &#8220;test run&#8221; to use the step-by-step wizard to pre-test the selected program. 
		Note: The robot does not run but rather the software tests the protocol for software problems. With established protocols, tapping, &#8220;skip setup&#8221; is acceptable.</li>
	</ol>
	</li>
	<li>Tap &#8220;Run Protocol&#8221;.</li>
	<li>When the dilution series is complete, re-cover the plate and place in a 37 &#176;C, 5% CO2, humidified incubator until the next feeding.</li>
	<li>Optional: Label the plate with the date, strain information, user name, media type and other pertinent information.</li>
	<li>Over the next several days, feed the 96-well plate as required using Protocol 4 below. 
	Note: A typical stem cell plate will require a full media change once every 24 hr and the SCGM does not contain Y27632 for feeding; only during the initial seeding after passage. Prior to feeding, check the plate for contamination and colony density. See step 3.16 for more on monitoring colony density.</li>
	<li>Over the next few days, several columns near the center of the plate (usually columns 5-8) will approach an ideal density for passage; to identify this density, compare the 96-well plate to the density range shown in Figure 3B with the following information in mind:
	<ol>
		<li>Passage when the space between the majority of the colonies is approximately 25% of adjacent colony diameter. 
		Note: The rationale to identify the time to passage is that another cycle of feeding and growth would result in colonies making contact, which should be avoided.</li>
	</ol>
	</li>
	<li>To start a uniformly diluted plate and begin regular culture, select a column that is at the ideal density and passage. See Protocol 5 to passage.</li>
</ol>
4. Feeding 96-well Plate Stem Cell Colonies
Note: The following protocol describes the feeding of one 96-well stem cell colony plate. The technique can be scaled up to accommodate 6 total plates in parallel.
<ol>
	<li>Remove the 96-well stem cell colony plate from the 37 &#176;C, 5% CO2, humidified incubator.</li>
	<li>Check the plate for contamination and cell density. In order to feed, ensure that the colony density is below the ideal passage density. See Figure 3B for information on density. 
	Note: Contamination may present as turbid media and be accompanied by an unpleasant smell. Small, obviously non-iPSC aggregates may also be visible under microscopic inspection. To evaluate cell density, see step 3.16.1 above.</li>
	<li>Place the 96-well stem cell colony plate in bed position 3 on a sloped, 37 &#176;C ramp.</li>
	<li>Load column 12 of the tip rack in bed position 1 with RT 200 &#181;l pipet tips.</li>
	<li>Place a 4-well trough in bed position 2.</li>
	<li>Fill well 3 of the trough in bed position 2 with 8 ml fresh, RT SCGM without Y27632 either by sterile pour or pipet transfer.</li>
	<li>Remove the 96-well stem cell colony plate lid placing it in the PLHR.</li>
	<li>Using the robotic control touch pad, press the following combination of buttons:
	<ol>
		<li>Tap &#8220;Run a Protocol&#8221;.</li>
		<li>Select &#8220;Colony Feed One Plate&#8221; and tap next.</li>
		<li>User decision: tap &#8220;test run&#8221; to use the step-by-step wizard to pre-test the selected program. 
		&#8203;Note, the robot does not run but rather the software tests the protocol for software problems. With established protocols, tapping, &#8220;skip setup&#8221; is acceptable.</li>
		<li>Tap &#8220;Run Protocol&#8221;.</li>
	</ol>
	</li>
	<li>When the feeding protocol is complete, re-cover the 96-well stem cell colony plate and return to the 37 &#176;C, 5% CO2, humidified incubator until the next feeding or passage is required. This is usually required after 24 hr. 
	Note: It is recommended to record the feeding event on the plate cover with the date, media type, user name and other pertinent information.</li>
</ol>
5. Passaging 96-well Plate Stem Cell Colonies
<ol>
	<li>After several feeding cycles the 96-well stem cell colony plate will be ready to passage. This protocol describes passage of one column to one 96-well plate, representing a 1:12 split ratio. The user can define the split ratio and select any number of wells or columns to split, including choosing wells that may not be adjacent.</li>
	<li>Compare the 96-well stem cell colony plate density to Figure 3B to determine whether to passage. The ideal passage density is when the space between the majority of colonies is approximately 25% of adjacent colony diameter or a subsequent feeding would result in the colonies growing into one another.</li>
	<li>Examine the 96-well stem cell colony plate under a microscope to ensure there is no contamination.</li>
	<li>Place the 96-well stem cell colony plate in bed position 3 on a sloped, 37 &#176;C ramp.</li>
	<li>Load columns 8-12 in the tip loading rack in bed position 1 with RT 200 &#181;l pipet tips.</li>
	<li>Place a 4-well trough in bed position 2.</li>
	<li>Leave trough well 1 empty, put 8.5 ml SCGM + 10 &#181;M Y27632 in well 2, put 3.5 ml 30% proteolytic and collagenolytic dissociation reagent (or EDTA based dissociation reagent) in well 3, put 4.5 ml PBS in well 4. 
	Note: All reagents can be at RT or 37 &#176;C. RT proteolytic and collagenolytic dissociation reagent at 30% diluted with PBS was used for the Adipose and Fibroblast derived iPSC culture described here.</li>
	<li>Place one new, pre-warmed 37 &#176;C extracellular matrix gel coated 96-well plate in bed position 5.</li>
	<li>Remove the 96-well stem cell colony plate lid, as well as the new 96-well plate lid and place them both in the PLHR.</li>
	<li>Using the robotic control touch pad, press the following combination of buttons:
	<ol>
		<li>Tap &#8220;Run a Protocol&#8221;.</li>
		<li>Select &#8220;Colony Split 1:12 Column 1&#8221; and tap next.</li>
		<li>User decision: Tap &#8220;test run&#8221; to use the step-by-step wizard to pre-test the selected program. 
		Note: The robot does not run but rather the software tests the protocol for software problems. With established protocols, tapping, &#8220;skip setup&#8221; is acceptable.</li>
		<li>Tap &#8220;Run Protocol&#8221;.</li>
	</ol>
	</li>
	<li>Once the passage protocol has finished, re-cover both plates.</li>
	<li>Optional: Label the new plate with the requisite information (i.e., date, cell line, passage number, media type, user name etc.).</li>
	<li>Return both plates to the 37 &#176;C, 5% CO2, humidified incubator until the next feeding or passage is required. Note: The next feeding is typically after 24 hr. 
	Note: Cells and colonies should attach to the plate within 2-3 hr of splitting and look very spread out. This is due to the presence of Rho-kinase inhibitor and the cells will condense and exhibit the characteristic stem cell morphology (i.e., cobblestone packed colonies with small cell volume to big nucleus ratio) after the first Rho-kinase inhibitor free feeding.</li>
</ol>
6. Harvesting 96-well Plate Stem Cells for Production
Note: Stem cell colonies may be harvested for use at any point during culture. Usually this occurs when the cells are ready to passage (see protocol 5). This protocol describes how to harvest eleven columns from a 96-well stem cell colony plate.
<ol>
	<li>Examine the 96-well stem cell colony plate under a microscope to ensure there is no contamination.</li>
	<li>Place the 96-well stem cell colony plate in bed position 3 on a sloped, 37 &#176;C ramp.</li>
	<li>Load columns 11 and 12 in the tip loading rack in bed position 1 with RT 200 &#181;l pipet tips.</li>
	<li>Place a 4-well trough in bed position 2.</li>
	<li>Leave trough well 1 empty, put 8.5 ml SCGM in well 2, put 3.5 ml 30% proteolytic and collagenolytic dissociation reagent (or EDTA based dissociation reagent) in well 3, put 4.5 ml PBS in well 4. 
	Note: All reagents can be at RT or 37 &#176;C.</li>
	<li>Remove the 96-well stem cell colony plate lid and place in the PLHR.</li>
	<li>Using the robotic control touch pad, press the following combination of buttons:
	<ol>
		<li>Tap &#8220;Run a Protocol&#8221;.</li>
		<li>Select &#8220;Colony Harvest Columns 2-12&#8221; and tap next.</li>
		<li>User decision: Tap &#8220;test run&#8221; to use the step-by-step wizard to pre-test the selected program. 
		Note: The robot does not run but rather the software tests the protocol for software problems. With established protocols, tapping, &#8220;skip setup&#8221; is acceptable.</li>
		<li>Tap &#8220;Run Protocol&#8221;. 
		Note: Once the collection procedure is complete, the cells will be in trough well 2 and ready for collection.</li>
	</ol>
	</li>
</ol>

The authors MKC, MJG, EEB, NJD and RO are or were at the time of development employees of InvivoSciences, INC which conceived of and developed the robotic protocols described here. TW is the CSO for InvivoSciences INC. SH is an employee of Gilson INC.

In the present study we developed a method of robotic culture for iPSC production that miniaturizes and automates feeding and passage in a 96-well plate format while also enabling efficient scale up. A liquid handling robot was used to routinely feed iPSC colonies and passage them at regular intervals by enzymatic dissociation. The robot was also programmed to produce extracellular matrix gel coated plates and perform a seeding density gradient. When fibroblast and adipose derived iPSCs were cultured in this system for greater than 20 passages, about 3 months, pluripotency was maintained, as were stable karyotypes. Both cell lines were capable of differentiation into cardiomyocytes. Together, the collection of protocols described here enables users with very little stem cell culture experience, or limited access to specialized culture equipment, to culture stem cells and achieve high cell production or multi-line handling with minimal labor and cost.
The robotic protocols detailed here provide a method for economical scale up of iPSC production and handling. One advantage is scalable stem cell culture in an established plate based format demonstrated here and previously44,47&#8211;49 to maintain pluripotency. While other formats of scalable stem cell culture yield pluripotent cells20,23,29 this plate based method requires essentially no change in culture format, like adaption to suspension, use of antifoaming chemicals or prolonged use of Rho-kinase inhibitor20. This plate based method can therefore rapidly, and predictably, accommodate new lines because the culture conditions are similar. As the use of iPSCs for disease modeling increases4,6,19,50, new iPSC lines are continuously generated. The techniques described here are best suited to culture new lines in medium to high volume and throughput because the barrier to transition onto the format is low. This is also true for the labor and equipment necessary to maintain the colonies. Finally, because the format and handling are similar to the environment used to derive and validate iPSC lines, culture performance, such as directed differentiation, is likely to be more predictable.
Figure 8 illustrates one strategy for scale up of stem cell production in 96-well plates. One plate (Cycle 0) is used to robotically seed 12 new plates, a 12 fold increase (Cycle 0 to 1). After several days of feeding, one of the twelve plates is passaged to seed another set of twelve plates (Cycle 1 to Cycle 2). The remaining eleven plates are now available for other uses and represent the production component of the system. At this rate, passage of one plate will provide 11 plates every cycle, or every 3-4 days. This methodology can be scaled more when multiple plates are passaged as shown in the transition from Cycle 2 to Cycle 3. In Cycle 3, two plates are always used to maintain the colony and seed 24 new plates. The remaining 22 plates are then available for use after every passage cycle. At any point in the maintenance cycle the user can seed more plates to increase production. One example would be to passage every column for two growth cycles. The first passage converts one plate into twelve plates, and each of the twelve plates is used to seed 144 plates. In this case, a user starts with one plate and after two passages, or about 1.5-2 weeks of culture, has 144 plates. For example, the fibroblast and adipose iPSC lines yield approximately 25-75 million cells per 96-well plate when ready to passage. 144 plates could therefore represent about 4-7 x 109 cells.
What is the labor burden for carrying 144 96-well plates robotically? One of the goals for this work was scalable stem cell production that reduced labor compared to traditional (i.e., 6-well plate) stem cell culture. The robot accomplishes this by alleviating the need to physically handle each plate during feeding and passaging. While 96-well plate production scales linearly like it would in traditional 6-well plates, the time a technician spends working with the plates is significantly less using robotic culture. The production efficiency of robotic culture is derived from this difference (Figure 9). It was found technicians could remove a 6-well plate from the incubator, check it on the microscope, then aspirate and feed the plate in approximately 3.5 minutes. In comparison, technicians spend approximately 30 sec&#160;removing a 96-well plate from the incubator and inspecting it on the microscope for density and contamination before placing it on the robot. With an expanded feeding protocol similar to the one described above, six 96-well plates can be loaded and fed, which takes approximately 21 min, or 3.5 min&#160;per plate. The total elapsed time to feed a plate is comparable between robotic and manual methods, but to hand feed 6 plates a technician is required for all 21 min&#160;whereas the technician spends only 3 min&#160;handling the six plates for the robot (a 30 sec&#160;inspection per plate). As Figure 9 shows, the time a technician spends handling 144 plates using the robot is about 1.2 hr as opposed to 8 hr manually and the robot will never make a mistake handling the plates or transferring liquids.
The 96-well iPSC culture methods described here provide a tractable platform for complex screening tasks. Because each well is genetically identical and seeded at the same density from the same initial pool, variables can be distributed across the plate and maintained by the robot with commercially available reservoirs to segregate conditions. This would be useful for experiments such as stem cell growth media development34,35,47, substrate or culture condition testing and toxicity studies utilizing stem cells51. Since each stem cell line is unique in terms of optimal colony size and passage requirements, one can use this system to modulate seeding density, feeding frequency and passage handling by manipulating the columns harvested, the mechanical agitation during passage and the feeding frequency. Iterating through these variables will allow the user to quickly find a set of conditions suitable for culture of a new line or develop new culture techniques. The robotic system is also capable of maintaining 96 parallel but independent stem cell colonies. When iPSC are derived from somatic cells or cloned after genetic manipulation, many parallel clones are screened to yield potential iPSC lines. Once single cells are seeded into 96-well plates, this system can feed and passage the 96-well plates keeping each colony segregated. This enables very high throughput when selecting potential clones and eliminates the difficulty in physically culturing 96 parallel lines. This method also allows scaled up production of each clone so that material is readily available for parallel analysis. Finally, we envision integrating these culture techniques with a robotic plate trafficking system that delivers plates to and from the incubator enabling fully automated culture. This could be accomplished with more complex robotics that allow for greater expansion since the basic protocols described here are transferable to other plate based systems.
The first critical steps to address in the protocols are ones that prevent and check for contamination such as steps 1.5 and 4.2. Contamination, as discussed above, can be successfully avoided if good sterile technique and common sense are utilized. It is imperative, regardless of stem cell culture format, that the operator check for contamination and doing so in this protocol at the indicated steps will significantly reduce the risk for contamination. Proper extracellular matrix gel application is a second essential step to the overall success of this protocol. Without properly coating the 96-well plates, the stem cells will not grow. One common issue is incomplete well coating. Experience has shown that air bubbles, electrostatic attraction and capillary action hinder complete well coating. The pre-wetting step (1.6) was developed to significantly improve well bottom coating and should not be skipped. This pre-wetting step appears to reduce the apparent hydrophobicity of the plastic 96-well plate such that when the extracellular matrix gel coating is applied it is evenly distributed across the well bottom and sides. It is also recommended to gently tap the 96-well plates against a clean hand once coated to ensure even extracellular matrix gel solution distribution. The dissociation parameters are also important to optimize. Extended incubation with enzyme or EDTA based dissociation reagent will result in single cells which may or may not be the goal during passage. Therefore, the operator should pay special attention to how dissociation time, trituration force, and wash repetitions affect colony morphology and health and adjust accordingly.
Understanding the limitations to the techniques described here are paramount to successful operation. As in any other cell culture setting, the use of 96-well plates presents a relatively high contamination risk. As described above, a technician is handling each plate during feeding and passaging; for example, when opening the lid, putting the plate on the robot bed, and checking the plate on the microscope. Therefore, as noted after step 1.5, it is imperative not to touch the inside of any plate lid or expose this part to any potentially contaminated surface such as the sloped heating blocks or vertical pins on the bed. During protocol development this limitation was discovered and was the impetus to develop a rack to hold plate lids to maintain sterility. Likewise, the trough reservoirs, pipet tips and other supplies on the liquid handling robot bed are potential sources of contamination because they are open to environment and handled by the technician. Therefore, good sterile technique should be applied such as reducing movements over exposed culture materials or open liquids. Another limitation is that when the protocol is scaled up, visually checking every well of &#62;100 96-well plates is cumbersome. This can be dealt with by visually inspecting the whole plate for signs of contamination, such as turbid media, to identify suspicious wells. For future scale up, use of an automated microscope and indicator of cell growth and potential contamination will be incorporated.
In summary, the high-throughput 96-well based platform described here offers a reproducible, high fidelity method for stem cell culture and production in a plate format. This method reduces the requisite experience, equipment necessary and labor time dedicated to stem cell culture while maintaining the benefits of traditional adherent culture.

The use of human induced pluripotent stem cells (iPSCs) has increased significantly since their derivation in 20071 for compound testing, regenerative medicine and disease modeling2&#8211;6. This demand comes from iPSC&#8217;s capacity to yield large numbers of pluripotent cells that can be differentiated into somatic cells at an unparalleled quantity. As directed differentiation techniques improve7&#8211;10 and development of human cell or tissue modeling and cell therapy increases11&#8211;14, so does the requirement for mass-production of high quality iPSCs. It is widely cited that among other maladies, myocardial infarction or b-cell functional replacement will require hundreds of millions to billions of iPSC derived somatic cells15&#8211;18. Furthermore, increasingly complex 3D tissue modeling for drug discovery and therapy will demand large numbers of cells9,13,19. In all of these examples, defined, uniform and reproducible iPSCs must be available and straightforward to produce.
In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Scaled production of iPSCs has focused on suspension culture20&#8211;25 or suspension with the use of microcarrier substrates26&#8211;28 partly because these techniques are successfully deployed for large scale, non-iPSC, eukaryotic based production. Several groups have demonstrated suspension culture systems that yield pluripotent stem cells20,21,23,24,29. However, these approaches utilize expensive and complex systems not readily available to researchers developing novel differentiation programs, driving tissue engineering and doing laboratory scale research. Furthermore, suspension and microcarrier iPSC cultures require adaptation and techniques or chemicals not found in traditional adherent iPSC culture such as continuous use of Rho-kinase inhibitor, antifoaming agents and filtration to regulate aggregation. Physical stress to the cells is more prevalent in suspension culture, introduced by mechanical agitation as well as during microcarrier collision. These issues limit the predictability and speed at which newly generated stem cell lines can be cultured in suspension. Others have developed robotic plate handling systems that mimic plate culture techniques30,31 but these platforms demand significant investments for equipment and expertise to operate in addition to the fundamental challenges associated with stem cell culture.
The following work describes the development and practice of a scalable iPSC culture system that utilizes a self-contained, standard 8-channel robotic liquid handler and 96-well plates. This method was designed to bridge laboratory scale iPSC culture and high-volume iPSC production (e.g., 107 &#8211; 1.5 x 109 cells per week per technician) enabling those developing new iPSC technologies to easily scale up production without large hardware or labor investments. This method is relatively inexpensive to set up, requires little to no stem cell culture, programming or engineering experience, has a small equipment footprint without the need for a dedicated cell culture hood, and utilizes standard cell culture equipment to enable medium to high throughput automated cell production. The aim was to develop a system capable of scalable stem cell culture that can be utilized by laboratories new to stem cell culture, those who find manual cultivation a barrier to developing their ideas or those wanting an economical means to produce large numbers of stem cells. The platform presented here removes technician influence on stem cell culture and normalizes the feeding and passage procedures to enable consistent stem cell production.

<table><tbody><tr><td>96-well plates</td><td>Corning</td><td>3596</td><td>96-well; Well volume: 360&amp;nbsp;&amp;#956;l; Cell growth area: 0.32&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;; Individually wrapped</td></tr><tr><td>Seahorse Trough</td><td>SeahorseBio</td><td>201308-100</td><td>Reservoir 4 Clear Part Poly Proplene 73Ml 25/Cs</td></tr><tr><td>Gilson Tips</td><td>Gilson</td><td>F167023</td><td>10 racks gilson 96 tips D200 tips</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>12500062</td><td>DMEM/F12 powder.&amp;nbsp; Resuspend in 1 L purified, cell culture grade water and sterile filter.</td></tr><tr><td>Growth Factor Reduced Matrigel</td><td>Corning</td><td>354230</td><td>Referred to as, &quot;extracellular matrix gel&quot; in the text. Matrigel GFR, 10&amp;nbsp;ml</td></tr><tr><td>mTeSR1</td><td>StemCell Technologies</td><td>5857</td><td>mTeSR1 Complete Kit for hES Maintenance.</td></tr><tr><td>E8 Media</td><td>StemCell Technologies</td><td>5940</td><td>TeSR-E8 Kit for hESC/hiPSC Maintenance</td></tr><tr><td>Y27632</td><td>AdooQ BioScience</td><td>A11001-50</td><td>Rock inhinitor Y-27632 2HCI</td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>ACCUTASE</td><td>Referred to as, &quot;proteolytic and collagenolytic dissociation reagent&quot; in the text. Accutase 500&amp;nbsp;ml sterile cell solution</td></tr><tr><td>PBS</td><td>Fisher</td><td>SH30256FS</td><td>PBS w/o Ca Mg 500&amp;nbsp;ml, 6/pk</td></tr><tr><td>Gilson PIPETMAX</td><td>Gilson</td><td>PIPETMAX</td><td>http://www.gilson.com/en/AI/Products/13.290/Default.aspx#.VCGwRBZmYSk</td></tr></tbody></table>

scalable 96-well plate based ipsc culture and production using a robotic liquid handling system

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Watch this Scientific Journal Video about Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System at JoVE.com

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

scalable-96-well-plate-based-ipsc-culture-production-using-robotic

Research

JoVE Journal

Developmental Biology

31.6K Views.  InvivoSciences, Inc.. The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes. 

Video: Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

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 ...

Bioengineering

Reusable Single Cell for Iterative Epigenomic Analyses

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. 
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. 
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic ...

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.

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 ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Biology

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Induced Pluripotent Stem Cell Differentiation Using an Automated Culture System

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Neuroscience

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Samenvatting

Meer video's verkennen