This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2EDK - 00474). We would also like to express our gratitude to the Choose Life Foundation for providing continuous support to the GMP Lab &#34;Dimitris Lois&#34; of the Institute of Cell Therapy.

This study was approved by the Ethics Committee of the University of Patras and the Institutional Review Board of the University General Hospital of Patras. Prior to obtaining biological specimens, informed consent was obtained from healthy individuals. The eligibility assessment of participants was conducted according to institutional procedures and in compliance with JACIE standards for leukapheresis. If this protocol is to be implemented in a clinical ACT setting, all procedures must adhere to Good Manufacturing Practice (GMP) guidelines and be performed in GMP-compliant clean rooms. Special safety precautions are necessary when working with viral vectors, and these procedures should be conducted within a dedicated biosafety cabinet. In this study, the described procedures were conducted within a validated H35 HEPA Hypoxystation (see Table of Materials) to ensure a controlled and monitored environment. Decontamination of all hazardous liquids should be carried out using 10% bleach solution.
1. PBMC isolation and T cell activation (Day 0)
<ol>
	<li>Collect cells from the donor apheresis bag (leukopaks, see Table of Materials) into 50 mL tubes, depending on the initial volume.</li>
	<li>Perform two washes of the cell bag by infusing 15 mL of complete hematopoietic media (+5% HSA, commercial serum-free hematopoietic media, see Table of Materials).</li>
	<li>Conduct polysaccharide-based density gradient centrifugation at a 1:2 (reagent:sample) dilution. Centrifuge at 800 x g for 30 min at room temperature with breaks off (A/D, 9/0).</li>
	<li>Using a micropipette, collect PBMCs into a clean 50 mL tube.</li>
	<li>Wash the cells twice with 1x PBS, filling up to 50 mL for each wash (400 x g, 10 min, room temperature). Resuspend the cells in 5 mL of complete media.</li>
	<li>Count the cells using trypan blue using either a standard hemocytometer or an automated cell counter (see Table of Materials).</li>
	<li>Dilute the cells with complete media to achieve a concentration of 2&#160;x 106 cells/mL.</li>
	<li>Activate 20 x 106 PBMCs by adding 10 &#181;L of T cell stimulation reagent (see Table of Materials) per 2 x 106 starting cells. 
	NOTE: This protocol is designed for the activation of 20 x 106 PBMCs but can be adjusted for larger cell numbers as needed.</li>
	<li>Add rhIL-2 (20 U/mL) (see Table of Materials), mix gently, and transfer the mixture into an appropriate vessel (e.g., 6-well plate).</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 for 72 h.</li>
</ol>
2. Lentiviral T cell transduction (Day 3)
<ol>
	<li>Transfer the cell culture into a suitable vessel, such as a 50 mL conical tube. Mix well and centrifuge at 300 x g for 10 min at room temperature (RT) to remove the activation reagent. Carefully discard the supernatant completely&#160;and resuspend the pellet in 1 mL of complete medium. 
	NOTE: At this stage, it is possible to thaw the virus on ice.</li>
	<li>Count the cells using trypan blue.</li>
	<li>Resuspend the cells in complete&#160;medium to achieve a concentration that allows for plating 0.5 x 106 cells in a total volume of 400 &#181;L per well in a 24-well plate. Add rhIL-7 (155 U/mL) and rhIL-15 (290 U/mL) (see Table of Materials). 
	NOTE: Ensure to account for the volume of viruses to be added. Refer to step 2.4.</li>
	<li>In a separate tube, add Vectofusin-1 (see Table of Materials) to Opti-MEM medium at a concentration of 10 &#181;g/mL, with a volume equal to the viral volume to be used. 
	NOTE: Consider the total cell culture volume when calculating the concentration of Vectofusin-1. See step 2.3. Alternatively, Vectofusin-1 GMP can be used as a GMP-grade reagent.</li>
	<li>Combine the concentrated virus at a Multiplicity of Infection (MOI) of 40 with the Vectofusin-1/Opti-MEM mixture (1:1 ratio) and mix thoroughly. Add this mixture to the cell culture and adjust the total volume to 400 &#181;L per well using complete medium, if necessary.</li>
	<li>Place the lid on the plate, seal it with paraffin film, and centrifuge at 1000 x g for 2 h at 32 &#176;C. Subsequently, incubate the plate at 37 &#176;C, 5% CO2 overnight.</li>
</ol>
3. Virus removal and T cell expansion (Day 4)
<ol>
	<li>Collect the cells and pool them into a suitable vessel, such as a 50 mL tube.</li>
	<li>Centrifuge the cells at 400 x g at room temperature (RT) for 5 min. Carefully remove the supernatant completely and resuspend the pellet in 1 mL of complete media.</li>
	<li>Count the cells using trypan blue. Resuspend the cells in complete media at a concentration of 1 x 106 cells/mL. Add rhIL-7 (155 U/mL) and rhIL-15 (290 U/mL) (see Table of Materials).</li>
	<li>Transfer the cells to an appropriate vessel, such as a T25 flask, at a concentration of 0.3-0.5 x 106 cells/cm2 and incubate them at 37 &#176;C, 5% CO2, for 2 days. 
	&#8203;NOTE: If a larger cell population is required, cells can be subcultured (1:2) in the presence of rhIL7 and rhIL-15 every 2 days, following the same incubation conditions mentioned above, until the desired cell numbers are achieved.</li>
</ol>
4. Cryopreservation (Day 14)
NOTE: Ice will be required to transfer cryovials to the storage location during this step. The timing of cryopreservation depends on the desired cell numbers. For this protocol, cryopreservation is performed on Day 14 after achieving a 25-fold expansion.
<ol>
	<li>Once the desired cell number is reached, harvest the cells and transfer them to 50 mL tubes (adjust the number of tubes based on the total volume).</li>
	<li>Centrifuge the tubes at 400 x g at room temperature (RT) for 5 min. Carefully remove the supernatant completely and resuspend the pellet in 10 mL of media. If necessary, pool the cells from multiple tubes.</li>
	<li>Count the cells using trypan blue. Keep an aliquot of 1 x 106 cells for each quality control (QC) testing (refer to Section 5) to assess Vector Copy Number and Transduction Efficiency.</li>
	<li>Centrifuge the tubes at 400 x g for 10 min at RT. At this point, label the cryovials accordingly.</li>
	<li>Completely remove the supernatant and resuspend the pellet in a cryopreservation solution consisting of 50% HSA and 40% HBSS at a concentration of 1 x 107 cells per mL per cryovial. Transfer the cell suspension into the appropriate number of cryovials and add 10% dimethyl sulfoxide (DMSO) to each cryovial.</li>
	<li>Place the cryovials on ice and promptly transfer them to a controlled-rate freezer for cryopreservation. After cryopreservation, transfer the cryovials to a monitored liquid nitrogen tank for long-term storage.</li>
</ol>
5. Quality control (Day 14)
NOTE: For release testing, the product underwent several tests, including sterility tests for microbial growth, and endotoxin level assessment. These tests were conducted in certified laboratories at the University General Hospital of Patras and the University of Patras. Mycoplasma presence was determined in-house using a biochemical detection assay and measured on a luminometer following the manufacturer&#39;s instructions (see Table of Materials). Cell phenotype (Figure 1) as well as cell number and viability were assessed in-house by flow cytometry (see section 5.1) and an automated cell counter&#160;(see Table of Materials), respectively. Release criteria were established based on published data and recommendations10,11for GMP-produced cell products (Table 1).
<ol>
	<li>Perform transduction efficiency-flow cytometry staining (Day 14) 
	NOTE: An aliquot of 1 x 106 cells will be stained for viability (7-AAD) and CD3 marker2 to evaluate the transduction efficiency (see Table of Materials). If the gene of interest, such as in the case of CAR T cells, needs to be detected, the specific antibody should be in a different color from that of the CD3 marker and 7-AAD to avoid compensation. Appropriate control samples should be taken into account.
	<ol>
		<li>Place the sample in a FACS tube and add 500 &#181;L of FACS buffer.</li>
		<li>Centrifuge the tube at 400 x g at RT for 5 min.</li>
		<li>Completely discard the supernatant with a pipette and resuspend the pellet in a 1:5 diluted solution of CD3:FACS buffer (see Table of Materials).</li>
		<li>Vortex the sample and incubate for 30 min on ice in the dark.</li>
		<li>Add 500 &#181;L of FACS buffer. Centrifuge at 400 x g for 5 min (RT).</li>
		<li>Completely discard the supernatant and resuspend the pellet in a 1:20 diluted solution of 7-AAD:FACS buffer (see Table of Materials).</li>
		<li>Vortex the sample and incubate for 10 min on ice in the dark.</li>
		<li>Add 200 &#181;L of FACS buffer and analyze the sample using a flow cytometer (see Table of Materials).</li>
	</ol>
	</li>
</ol>

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N.S. developed the protocol and wrote the manuscript which was reviewed and supervised by A.S.; K.S., G.A. performed additional experiments; D.K., M.L. provided valuable support in the experiments and along with V.Z., N.T. in the review process. N.T. is a member of ProtATonce Ltd. All other authors declare no competing interests.

Lentiviral vectors as gene delivery vehicles are important tools in the field of cell and gene therapy because of their ability to transduce both dividing and non-dividing cells12. However, large-scale production of lentiviral vectors using GMP-compatible methods is still challenging due to various parameters, such as transfection methods and purification steps, which can result in variability in lentivirus batches. Academic centers often obtain viral stocks from viral production biotechnology com...

The emergence of Adoptive Cell Therapies (ACT) and Chimeric Antigen Receptor (CAR)-T cells have brought about a revolutionary shift in modern clinical practice, establishing a new paradigm of personalized medicine. Particularly, CAR-T cells targeting CD19 have demonstrated exceptional clinical responses and represent the most advanced T cell therapy for B cell malignancies1,2,3,4,5. However, the current commercialized gene-modified T cell therapies heavily rely on viral vectors for gene transfer. The implementation of viral vectors in ACT, especially under Good Manufacturing Practice (GMP) conditions within an academic setting, presents significant challenges due to scalability limitations, specialized equipment requirements, the need for highly skilled personnel, and the use of GMP-compliant reagents for plasmid-mediated viral particle production6,7,8.
Consequently, the manufacturing process for gene-modified T cell therapies is intricate and typically commences with the isolation of Peripheral Blood Mononuclear Cells (PBMCs) from a leukapheresis product. T cells are often enriched from the PBMC pool and activated using anti-CD3/CD28 micro-complexes7,9. Subsequently, T cells are genetically modified with either viral or non-viral vectors, which can be produced in situ or outsourced. To attain the required cell numbers for infusion, gene-modified cells are expanded before final formulation and/or cryopreservation. Finally, the cell product must satisfy various release criteria based on multiple quality control assays9.
This protocol presents a comprehensive methodology utilizing simple techniques for the ex vivo manipulation of healthy PBMCs to manufacture a gene-modified T cell product under GMP-compliant conditions. The protocol incorporates the use of a lentiviral vector, which can be outsourced to a viral production company providing all necessary quantitative/functional, purity, and safety tests (e.g., viral titer, detection of host cell DNA replication-competent lentivirus). For this study, the vector employed is a VSV-g (Vesicular Stomatitis Virus G Protein)-based second-generation lentiviral vector with Green Fluorescent Protein (GFP), as the gene of interest, in constitutive expression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well TC-treated plate</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL FACS tube&#160;</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>Greiner biomedica</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well TC-treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>7-AAD</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS CANTO II</td>
			<td>BD Biosciences</td>
			<td>V96300084</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Applichem</td>
			<td>A1391</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter&#160;</td>
			<td>Corning Cell Cytosmart</td>
			<td>J21E0081</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Greiner biomedica</td>
			<td>122263</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Life Technologies</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Buffer (1x D-PBS, 0.5% BSA, 2 mM EDTA)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software v10.6.2&#160;</td>
			<td>TreeStar Inc</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco CTS Opti-MEM I Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A4124801</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-15</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-114</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-2</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-146</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-7</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>14175</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPA Whitley H35 Hypoxystation</td>
			<td>Don Whitley Scientific</td>
			<td>HA0315172H</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin (HSA)</td>
			<td>Baxter</td>
			<td>B05AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Junior LB 9509 Portable Luminometer</td>
			<td>Berthold Technologies</td>
			<td>6506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Provirus Quantitation Kit</td>
			<td>Takara Bio</td>
			<td>631239</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>7811</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP T cell TransAct</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-156</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human&#160; CD3 (Clone: UCHT-1)</td>
			<td>BD Biosciences</td>
			<td>557706</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Plus Detection kit</td>
			<td>Lonza Bioscience</td>
			<td>LT07-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm ultra-expandable closing film tape 5 cm x 75 m</td>
			<td>D.Dutscher</td>
			<td>90261</td>
			<td></td>
		</tr>
		<tr>
			<td>PeriStem PS-650-DB (apheresis bag)</td>
			<td>Biomed Device</td>
			<td>SC00816</td>
			<td></td>
		</tr>
		<tr>
			<td>Planer Kryo10 Series III</td>
			<td>Planer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Greiner biomedica</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectofusin-1 GMP</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-165</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVOTM 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza Bioscience</td>
			<td>A1048501</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Assessment of transduction efficiency 
The transduced cells were evaluated for the expression of the gene of interest (GFP) using flow cytometry. Representative results are depicted in Figure 1. On Day 14, more than 95% of the cells were CD3+, indicating successful T cell activation and expansion. The transduction efficiency within the CD3+ population was measured at 58.7% (TD, Figure 1C) compared to the non...

Watch this Scientific Journal Video about A GMP-Compliant Procedure for the Generation of Gene-Modified T cells at JoVE.com

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

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 ...

a-gmp-compliant-procedure-for-the-generation-of-gene-modified-t-cells

Research

JoVE Journal

Bioengineering

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

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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

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

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

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

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

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...