handling neuronal stem cell cultures in a cell production facility

Handling Neuronal Stem Cell Cultures in a Cell Production Facility

Place three Petri dishes in the water pan at the base of each incubator. Fill these dishes with sterile water, and maintain the water level in these dishes as they are critical for maintaining the relative humidity set point in the incubator.
Within the graphical interface, click on Incubator. Subsequently, click on the existing value for relative humidity under the set point and enter 85%. Then, click on the green check mark to accept this value.
Next, adjust the buffer chambers, the process chamber, and an incubator to the gas set points required for the type of cells being introduced. Clean the surface of the process chamber with sterile gauze, and a non-flammable disinfectant, such as benzalkonium chloride-based products.
Then, clean the gloves and surfaces that are commonly touched, such as door handles. Subsequently, clean the surface of the laminar flow hood and buffer chamber with 70% ethanol and allow it to dry.
After that, place the flask or plate of cells in the hood, and briefly wipe its exterior surface with a sterile gauze sponge saturated with ethanol. Then, put the flask or plate in the buffer chamber and run a dilution factor. Upon completion, immediately transfer the flask or plate to the appropriate incubator.
As the incubator is at the back of the process chamber, pull a shelf out into the process chamber for cell placement and avoid opening the incubator doors unnecessarily or for extended periods of time.
In this step, prepare the cell culture medium. Allow the medium to equilibrate with the carbon dioxide and oxygen levels present within the system by leaving the media container slightly uncapped for 20 minutes.
Afterward, remove the old medium from the wells. For pluripotent stem cells, remove almost all of the old medium with a small amount of the old medium remaining, to prevent desiccation during the feeding process. For neural stem cells, remove half of the old medium. Then, add fresh medium to the cell culture plates, and place the plates back into their designated incubator.

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1. Initial Setup
<ol>
	<li>Setting Gas and Temperature Concentrations
	<ol>
		<li>Using the software, click the &#34;Guest&#34; tab on the upper left corner of the graphical interface. Log in to the system using a designated username and password. Make sure each user has their username and password.</li>
		<li>Click on a module to adjust (Figure 1). Within the new window displaying the current settings, click on the existing O2 set point value below &#34;Set point&#34; and enter this module&#39;s required O2 concentration level. Enter 5% O2 if pluripotent stem cells will be grown or manipulated in this module and 9% O2 if neural stem cells will be grown or manipulated. Click on the green check mark to confirm the set point. 
		Note: Two modules are not gas-adjustable: the laminar hood and the microscope chamber. The gas concentrations in the laminar flow hood are atmospheric, while those in the process chamber passively maintain those in the microscope chamber.</li>
		<li>Repeat this step for CO2. Enter a set point of 5% CO2 for all modules except for the buffer chambers, which only adjust for O2.</li>
		<li>Monitor the current gas concentrations labeled &#34;process values&#34; to ensure they reach the new set points.</li>
		<li>Adjust the gas set points of all modules to the appropriate values. Match the CO2 and O2 levels of chambers that will be exposed to one another. For example, adjust the process chamber to 5% O2 before opening an incubator that grows cells at 5% O2. Also, change to 5% O2 in any buffer chamber used to add items to the process chamber during this time.</li>
		<li>Set the temperature of the process chamber to 37 &#176;C using the same screen for gas adjustments. Also, the process chamber floor temperature should be set to 37 &#176;C.</li>
		<li>Click on the incubation module banks underneath each incubator. Adjust the temperature of the banks to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Operation of Buffer Chambers
	<ol>
		<li>Gather all the supplies required for the given task (feeding, splitting, staining, etc.) in the beginning to prevent a delay in the workflow. Liberally spray all materials with 70% ethanol and allow them to dry in the laminar hood. Do not spray flasks or plates of cells&#8212;wipe them gently with a sterile gauze sponge saturated with 70% ethanol.</li>
		<li>Ensure the outer and inner doors of the buffer chamber are securely closed. Then, open the outside door and place the items inside.</li>
		<li>Close the outer door. Select the buffer module within the software and click on the dilution factor tab. Enter one into the log factor box. Click start. 
		Note: A &#34;dilution factor&#34; means that the buffer module&#39;s atmosphere will be evacuated and replaced once with clean, HEPA-filtered gas.</li>
		<li>Once the graphical interface stops flashing &#34;dilution factor,&#34; open the buffer chamber&#39;s inner door and bring the materials inside the process chamber. 
		Caution: Do not allow the inner and outer doors ever to be open simultaneously, and do not open the inner door if the buffer chamber has not undergone a dilution factor.</li>
		<li>First, remove items from the process chamber and ensure that the buffer chamber has undergone a dilution factor. Then, open the inner door, place the items to be removed inside, and close the inner door. Then, open the outer door and remove the items. 
		Note: A dilution factor must not be run before opening the outer door.</li>
	</ol>
	</li>
</ol>
2. Introducing and Feeding Cells
<ol>
	<li>Incubator Setup
	<ol>
		<li>Place 3 Petri dishes in the water pan at the base of each incubator. Fill these dishes with sterile water, not directly filling the pan. Maintain the water level in these dishes, as they are critical for maintaining the incubators&#39; relative humidity (RH) set point.</li>
		<li>Within the graphical interface, click on the incubator. Under the set point, click on the existing relative humidity (RH) value and enter 85%. Click on the green check mark to accept this value.</li>
	</ol>
	</li>
	<li>Preparation of the Cell Production Facility for Cells
	<ol>
		<li>If not already done so, adjust the buffer chambers, the process chamber, and an incubator to the gas set points required for the type of cell being introduced.</li>
		<li>Clean the surface of the process chamber with sterile gauze and a non-flammable disinfectant. Do not use any peracetic acid-based disinfectant; in a closed system, the strong, lingering vapors are toxic to mammalian cells. Instead, use benzalkonium chloride-based products.</li>
		<li>Continue disinfecting. Clean the gloves and surfaces commonly touched, such as door handles. Use the disinfectant thoroughly but sparingly, as excess liquid in the process chamber will contribute to humidity, condensation, and possible microbial growth.</li>
		<li>Clean the surface of the laminar flow hood and buffer chamber with 70% ethanol and allow it to dry. Place the flask or plate of cells (in our case, PSCs(pluripotent stem cells) or NSCs(neural stem cells)) in the hood and briefly wipe its exterior surface with a sterile gauze sponge saturated with ethanol.</li>
		<li>Put the flask or plate in the buffer chamber and run a dilution factor (step 1.2.3).</li>
		<li>Upon completion of the dilution factor, immediately move the flask or plate to the appropriate incubator. As the incubators are at the back of the process chamber, pull a shelf out into the process chamber for cell placement. Avoid opening the incubator doors unnecessarily or for extended periods, as the process chamber&#39;s air is very dry compared to that of the incubators and will result in a significant loss of humidity in the incubator.</li>
	</ol>
	</li>
	<li>Feeding Cell Cultures
	<ol>
		<li>Gather medium components, such as serological pipettes, an electronic pipettor, gauze, and disinfectant. Also, gather a waste container for the spent cell culture medium, but do not fill it with bleach. Bring the materials into the process chamber through a buffer chamber as described in Section 1.2. Arrange materials to enable an optimal working space. Avoid placing items not being used in the center of the process chamber.</li>
		<li>Leave the media container slightly uncapped to allow the medium to equilibrate with the CO2 and O2 levels present within the system. Some PSC medium manufacturers indicate not to warm media at 37 &#186;C; if so, use a buffer chamber to equilibrate the medium. Allow the medium to equilibrate for 20 min.</li>
		<li>Remove the old medium from wells using an appropriate stereological pipette and an electronic pipettor. For PSCs, remove all but a small remaining amount of the old medium to prevent desiccation during the feeding process. For NSCs, remove half of the old medium. Pipet the waste into the waste container.</li>
		<li>Place the used pipette back into its original wrapper and move it to the side of the process chamber or into a buffer chamber designated for waste removal. With a fresh sterile serological pipette, add fresh medium to cell culture plates and place the plates back into their designated incubator.</li>
		<li>At the end of feeding, spray gauze with disinfectant and thoroughly clean the floor and door handles of the process chamber. If multiple cell lines are being grown in the system, sterilize between feeding each cell line. This helps minimize the risk of cross-contamination.</li>
		<li>Remove waste or other unneeded items through one of the buffer chambers upon completion.</li>
	</ol>
	</li>
</ol>
3. Splitting Cell Cultures
<ol>
	<li>Enzymatic Passaging of PSCs or NSCs
	<ol>
		<li>Gather all items needed for passaging. This includes media, enzyme or non-enzymatic cell dissociation buffer (the latter is for NSCs only), DPBS (Dulbecco&#39;s Phosphate Buffered Saline), plates or flasks, extracellular matrix (ECM), conical tubes, and serological pipettes. Bring them into the system using a buffer chamber.</li>
		<li>Coat fresh plates or flasks with ECM, as previously described 9,10. Some ECMs -such as those derived from mouse sarcoma cell lines - are only liquid between 4 and 15 &#176;C, so use a sterilized cold block or gel ice pack to keep the ECM cold while working in the heated process chamber.</li>
		<li>Pipet off the spent medium from the culture and dispose of it in the waste container.</li>
		<li>Rinse the well or flask surface using 1 ml of DPBS/ well and dispose of the DPBS.</li>
		<li>Add 1 - 2 ml of warm enzyme to each well. Only very dense cultures require 2 ml.</li>
		<li>Immediately move the culture dish or flask to the microscope chamber and observe the culture. Watch for signs of individual cells beginning to loosen off the dish. Do not wait until the cells float into suspension before proceeding to the next step.</li>
		<li>Return the cells to the main process chamber. Using a 5 ml serological pipette, add 4 ml of DPBS for each 1 ml of enzyme, and then forcefully pipet up and down to dislodge the cells from the well surface. If passaging multiple wells within a multiwall plate, add the DPBS to each well before dislodging the cells from the individual wells.</li>
		<li>Transfer the enzyme and PBS cell suspension to an appropriately sized conical tube.</li>
		<li>If a centrifuge is unavailable in the cell production facility, tightly seal the conical tube, place it in a buffer chamber, and seal the door closed.</li>
		<li>Remove the conical tube from the buffer chamber from the laminar flow side and spin the cells at 100 x g for 5 min at RT.</li>
		<li>Spray the conical tube with 70% ethanol and bring it into the process chamber using a buffer chamber and dilution factor.</li>
		<li>Pipet off the supernatant and resuspend the cells in 2 ml of PSC or NSC medium. If passaging PSCs, include ROCK(Rho-associated protein kinase) inhibitor in the medium at a concentration of 10 &#181;M.</li>
		<li>Count the cells using a hemocytometer and determine the wells or plates required. Plate PSCs at 5 x 104 - 1 x 105 cells/cm2. Split the NSCs at a 1:2 ratio. 
		Note: NSCs often clump and resist accurate counting in a hemocytometer.</li>
		<li>If cryopreservation of the cells is required, ensure that all supplies and freezing media are prepared and placed inside the process chamber. DMSO(Dimethylsulfoxide) is very toxic to cells at 37 &#186;C, so it works quickly. Keep the isopropanol-jacketed freezing container at RT in the laminar flow hood. Once the cryopreservation vials are filled and sealed, immediately remove them from the process chamber by passing them through a buffer chamber.</li>
	</ol>
	</li>
	<li>Manual Passaging of PSCs
	<ol>
		<li>Confirm that the culture needs to be passed, as previously described. If so, prepare fresh plates or flasks coated with extracellular matrix. Then, change the medium entirely.</li>
		<li>Clean the microscope chamber with sterile gauze moistened with a sparing amount of non-flammable disinfectant&#8212;too much will result in excess fogging. Only clean the gloves, floor area, and stage. Bring several 200 &#181;l or 1,000 &#181;l individually wrapped pipet tips into the chamber.</li>
		<li>Bring the plate of PSCs into the microscope chamber, remove the lid, and place it face down on the freshly cleaned floor. Leaving the lid up exposes the underside directly to air currents and potential contamination.</li>
		<li>Unwrap a pipet tip, and using the microscope to visualize the culture, pick apart colonies with good morphology using the tip of the pipet. 
		Note: The pipet tip may be attached to a pipet if the individual user finds this more comfortable.</li>
		<li>Replace the lid on the plate and move the plate back to the process chamber.</li>
		<li>Pipet off excess ECM from the new plate and transfer the media from the old plate (containing the colony pieces in suspension) to the new plate. If the old plate still contains colonies that are not ready to be picked, feed them as well.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Xvivo System</td>
			<td>Biospherix</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Xvivo Software</td>
			<td>Biospherix</td>
			<td>version i.o.2.1.2.1</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 Manifold</td>
			<td>Amico</td>
			<td>P-M2H-C3-S-U-OXY</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Manifold</td>
			<td>Amico</td>
			<td>M2H-C3-D-U-CO2</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Manifold</td>
			<td>Western Innovator</td>
			<td>CTM75-7-2-2-BM</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 Gas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Gas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Gas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>BDH</td>
			<td>BDH1164-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>benzalkonium chloride-based disinfectant&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s-based Cell dissociation Buffer</td>
			<td>Life Technologies</td>
			<td>13150-016</td>
			<td></td>
		</tr>
		<tr>
			<td>2mL Serological pipet</td>
			<td>VWR</td>
			<td>89130-894</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-102</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-104</td>
			<td></td>
		</tr>
		<tr>
			<td>25mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-106</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Serological pipet</td>
			<td>Olympus Plastics</td>
			<td>12-107</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>20uL pipet tips</td>
			<td>Eppendorf</td>
			<td>22491130</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL pipet tips</td>
			<td>Eppendorf</td>
			<td>22491148</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 pipet tips</td>
			<td>Eppendorf</td>
			<td>22491156</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope with DP21 camera and fluorescence</td>
			<td>Olympus Corporation</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Sodium</td>
			<td>Hyclone</td>
			<td>9236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Protide</td>
			<td>PP1130</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Stover, A. E., et al,. <a href="https://app.jove.com/v/53685">Culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research.</a> J. Vis. Exp. (2016).
The video outlines the procedure for maintaining a controlled environment in a cell culture workstation for the growth and maintenance of neuronal stem cells. These procedures involve setting chamber conditions, cleaning protocols, and media preparation, Thus, maintaining maximum sterility and reproducibility and replacing the traditional methods.

<img alt="Figure 1" src="/files/ftp_upload/22542/22542_Figure1.jpg" /> 
Figure 1. Graphical Interface for the Cell Production Facility. (A) The graphical representation of the CPF is the default screen and matches the CAD drawing shown in Figure 3. A mouse click over Buffer Module 1 opens its control screen (B), where O2 values are set to match the processing chamber. Similarly, clicking the Process Chamber or Incubator 1 ...

1. Initial Setup

	Setting Gas and Temperature Concentrations
	
		Using the software, click the &#34;Guest&#34; tab on the upper left corner of the ...

To minimize contamination in neuronal stem cell cultures, use a facility with a laminar hood, a buffer chamber, a process chamber, and a microscope chamber, each separated by airtight doors.
Now, fill water into a culture plate in the incubator to maintain the humidity.
Adjust the buffer chamber, the process chamber, and the incubator parameters to optimum levels.
Clean all surfaces with a disinfectant and sterile gauze to eliminate microbial contaminants.
Place the neuronal stem cell culture plate first in the laminar hood that provides filtered air.
Wipe the outside of the plate with ethanol-soaked gauze and then transfer it to the buffer chamber that provides filtered air with optimum gas levels.
Finally, move the plate through the process chamber into the incubator and close the door.
After incubation, move the culture plate back to the process chamber along with some fresh medium.
Equilibrate the fresh medium.
Refresh the medium in the culture plate and re-incubate the plate.

8 Views. Handling Neuronal Stem Cell Cultures in a Cell Production Facility

Video: Handling Neuronal Stem Cell Cultures in a Cell Production Facility - Experiment

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 ...

JoVE Journal

Bioengineering

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Handling Neuronal Stem Cell Cultures in a Cell Production Facility

Transcript

Handling Neuronal Stem Cell Cultures in a Cell Production Facility