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EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Splenectomy and T Cell Preparation
Day 0:
<ol>
	<li>Pour 10 ml of T cell media (TCM) into a 50 ml conical and place on ice for spleen collection.</li>
	<li>Sacrifice the appropriate number of animals by CO2 asphyxiation and secondary decapitation: Place animals in a cage receiving CO2 at a flow rate of 10-30% cage volume/minute, per American Veterinary Medical Association guidelines (no more than 5 animals can be sacrificed simultaneously) until respiration terminates and for two minutes thereafter. Remove animals from the CO2 chamber and decapitate. 
	NOTE: One spleen of a 6&#8211;12-week-old female C57BL/6 mouse will yield approximately 4.5-5 x 107 splenocytes. Here, 4 spleens will be harvested for approximately 200 x 106 cells.</li>
	<li>Lay the mouse such that its right side is facing up, and spray with 70% ethanol. With the forceps, grab a thin fold of skin below the left ribcage and cut a slight incision with the scissors. Peel back the skin, carefully grab a thin fold of the peritoneum with forceps, and cut a small cavity.</li>
	<li>The spleen is a small, elongated, dark red organ that resembles a flattened bean; delicately grab the spleen with the forceps and excise by cutting away the surrounding connective tissue. Place the excised spleens into the conical containing 10 ml of TCM on ice.</li>
	<li>Pour spleens over a 70 &#956;m mesh cell strainer and disaggregate by mashing with the blunt end of the inside of a 5 ml syringe to generate a single-cell suspension. A maximum of two spleens should be disaggregated per mesh strainer.</li>
	<li>Use a small volume of TCM to carefully wash the strainer following disaggregation to collect any remaining splenocytes. Pool all disaggregated spleens into a single-cell suspension. Bring final volume to 50 ml with TCM for one wash and spin at 300 x g for 10 min.</li>
	<li>Prepare a solution of 1x lysis buffer by adding 5 ml 10x lysis buffer to 45 ml sterile water. To eliminate red blood cells, resuspend pellet in 5 ml of 1x lysis buffer per spleen in a 50 ml conical, mixing well by gently pipetting up and down. If exceeding 5 spleens, use a 250 ml centrifuge tube. Place conical or centrifuge tube in a 37 &#176;C water bath for 5 min.</li>
	<li>Remove the lysis reaction from the water bath and add TCM at a 1:1 ratio with lysis buffer to neutralize the reaction. Wash by spinning at 300 x g for 10 min.</li>
	<li>Aspirate supernatant and fully resuspend pellet in TCM (2 ml/spleen, 8 ml total for 4 spleens) by pipetting up and down.</li>
	<li>Count cells by adding 10 &#956;l of cell suspension to 190 &#956;l trypan blue (1:20 dilution). Multiply the number obtained in one of the four gridded squares by 20 x 104 x total volume (8 ml) to obtain the total cell number (e.g., 125 cells x 20 x 104 x 8 ml = 200 x 106 splenocytes).</li>
	<li>Dilute cells to a concentration of 2 x 106 cells/ml in TCM, supplemented with 2 &#956;g/ml concanavalin A (ConA) and 50 IU/ml recombinant human interleukin-2 (rhIL-2). Thus, for 200 x 106 splenocytes, add 92 ml media to 8 ml cells, 200 &#956;g ConA, and 5,000 IU rhIL-2.</li>
	<li>Add 2 mL cells to each well of 24-well tissue-culture treated plates, such that 4 x 106 cells are in each well (e.g., 100 ml of cells will require approximately 4 plates).</li>
	<li>Incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
2. Transduction
Day 1:
<ol>
	<li>Calculate the number of non-tissue culture treated 24-well plates needed for transduction by multiplying the number of spleens harvested by 2 (8 plates are needed for 4 spleens).</li>
	<li>Next, calculate the required volume of recombinant human fibronectin fragment (RHFF) solution needed to coat plates by multiplying (total number of wells + 3) x 0.5 ml (8 plates x 24 wells = 192 + 3 = 195) x 0.5 ml = 97.5 ml.</li>
	<li>Prepare 97.5 ml phosphate-buffered saline (PBS) containing RHFF at a concentration of 25 &#956;g/ml by multiplying the total volume by 25 &#956;g (97.5 x 25 = 2437.5 &#956;g). Add this amount of RHFF to 97.5 ml PBS.</li>
	<li>Coat non-tissue culture treated 24-well plates by adding 0.5 ml PBS/RHFF solution per well.</li>
	<li>Incubate overnight at 4 &#176;C.</li>
</ol>
Day 2:
<ol start="6">
	<li>Dump PBS/RHFF solution from non-tissue culture treated 24-well plates.</li>
	<li>Add 1 ml/well 2% bovine serum albumin (BSA) in PBS and incubate at room temperature for 30 min.</li>
	<li>Remove BSA by firmly upending plate. Wash by adding 2 ml PBS.</li>
	<li>Collect viral supernatant by transferring media from each Human embryonic kidney 293T (HEK293T)-poly-D-lysine (PDL) coated plate into a 250 ml centrifuge tube and spin 10 min at 500 x g.</li>
	<li>Carefully transfer viral supernatant into a fresh 250 ml centrifuge tube, being sure not to disturb the cell pellet that may have formed.</li>
	<li>Add fresh TCM to the viral supernatant such that the final volume is 3 ml more than the amount needed for RHFF-coated wells. For example, for 192 RHFF-coated wells, bring the volume of viral supernatant to 192 + 3 ml = 195 ml.</li>
	<li>Remove cultured splenocytes from the incubator and resuspend by gently pipetting up and down 2 - 3 times in each well. Transfer to a 250 ml conical. If using a multichannel pipette, using a sterile reservoir prior to transfer will help expedite this step. Count as previously described and spin at 300 x g for 10 min.</li>
	<li>Add recombinant human interleukin-2 (rhIL-2) to viral supernatant at a concentration of 50 IU/ml. For example, add 50 x 195 = 9,750 IU rhIL-2 to 195 ml of viral supernatant.</li>
	<li>Resuspend splenocytes in viral supernatant at 1 x 106 cells/ml</li>
	<li>Add 1 ml/well of splenocyte suspension to RHFF-coated 24-well plates.</li>
	<li>Spin for 90 min according to the following settings: 770 x g, acceleration = 4, brake/deceleration = 0, 32 &#176;C.</li>
	<li>Prepare a TCM solution with 50 IU/ml rhIL-2. For example, prepare a 200 TCM solution by adding 10,000 IU rhIL-2. Add 1 ml of rhIL-2/TCM to each well after centrifugation.</li>
	<li>Culture overnight at 37 &#176;C in 5% CO2.</li>
</ol>
3. CAR T cell Culture and Harvest
Days 3 and 4:
<ol>
	<li>If T cells achieve &#62;80% confluence, cells may be split (this usually occurs by day 3 or day 4). To split cells, gently pipet up and down in each well 2-3 times, and move 1 ml from each well into new wells of a fresh 24-well tissue-culture treated plate. Then, add 1 ml of fresh TCM with 50 IU/ml IL-2 to each well such that final volume is 2 ml in all wells.</li>
	<li>If cells do not reach &#62;80% confluence, perform a half media change by slowly pipetting off 1 ml of media from the top of each well. Avoid disturbing cells settled on the bottom while removing media. Add 1 ml of fresh TCM containing 50 IU/ml rhIL-2.</li>
</ol>
Day 5:
<ol start="3">
	<li>Resuspend CAR T cells by gently pipetting up and down 3 times in each well and transfer to a 250 ml centrifuge tube. Spin cells at 300 x g for 10 min.</li>
	<li>Completely aspirate supernatant without disturbing pellet. Wash once with PBS, count cells as previously described, and wash with PBS a second time. A typical CAR T cell yield is approximately 1 x 106 cells per well (8 plates x 24 wells = 192 x 106 CAR T cells).</li>
	<li>Resuspend cells in PBS at a concentration of 5 x 107/ml for an injection of 1 x 107 in a volume of 200 &#956;g (e.g., for 192 x 106 CAR T cells, resuspend washed pellet in 3.84 ml PBS).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Concanavalin A</td>
			<td>Sigma Aldrich</td>
			<td>C2010</td>
			<td>Non-specific mitogen to induce T cell proliferation and viral transduction</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>T cell culture media</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), Fraction V&#8212;Standard Grade</td>
			<td>Gemini Bio Products</td>
			<td>700-100P</td>
			<td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td>
		</tr>
		<tr>
			<td>Pharm Lyse (10X concentrate)</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td>Lyses red blood cells during splenocyte processing</td>
		</tr>
		<tr>
			<td>70 &#181;m Sterile Cell Strainers</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Filters away large tissue particles during splenocyte processing</td>
		</tr>
		<tr>
			<td>100 mm BioCoat Culture Dishes with Poly-D-Lysine</td>
			<td>Corning</td>
			<td>356469</td>
			<td>Promotes HEK293 cell adhesion to maximize proliferation after transfection</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Life Sciences</td>
			<td>D2650</td>
			<td>Necessary for complete dissolution of temozolomide</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Hospira</td>
			<td>IM 0132 (5/04)</td>
			<td>Solvent for temozolomide and ketamine/xylazine</td>
		</tr>
		<tr>
			<td>Ketathesia HCl</td>
			<td>Henry Schein Animal Health</td>
			<td>11695-0701-1</td>
			<td>Ketamine solution</td>
		</tr>
		<tr>
			<td>AnaSed</td>
			<td>Lloyd Inc</td>
			<td>N/A</td>
			<td>Xylazine sterile solution 100 mg/mL</td>
		</tr>
	</tbody>
</table>

an ex vivo technique to generate tumor-specific chimeric antigen receptor t cells

Source: Riccione, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52397">Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care.</a> J. Vis. Exp. (2015).
This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host genome and produce surface-bound CARs targeting the tumor antigen.

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take retroviral vectors mixed with primary spleen cells.
The vectors contain transgenic RNA encoding a chimeric antigen receptor or CAR, which targets a tumor-specific antigen.
The cytokine interleukin-2 in the media selectively promotes T cell proliferation while the other cells eventually die.
Transfer the mixture to a microplate coated with recombinant fibronectin fragments.&#160;
Centrifuge to facilitate the fibronectin fragments binding the vectors and T cells, mediating virus-host cell interaction, and incubate.
Upon fusion of the vectors&#39; envelope with the cell membrane, the viral enzymes reverse-transcribe the released RNA into DNA and incorporate it into the host genome.
The transduced T cells express surface-bound CARs, consisting of an extracellular antibody variable fragment targeting the tumor-specific antigen and intracellular signaling regions to respond to the antigen.
Resuspend the cells before transferring them to a tube.
Centrifuge to discard the supernatant containing non-internalized viruses, and resuspend the cells in a buffer for downstream assays.

an-ex-vivo-technique-to-generate-tumor-specific-chimeric-antigen

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

68 vistas. Fuente: Riccione, K., et al. Generación de células CAR-T para la terapia adoptiva en el contexto del glioblastoma estándar de atención. J. Vis. Exp. (2015). Este video muestra una técnica para la generación ex vivo de células T con receptor de antígeno quimérico (CAR) específico de antígeno tumoral. Los vectores retrovirales portadores de ARN transgénico se mezclan con células de bazo en un medio suplementado con interleucina-2 para promover la supervivencia y proliferación de las...

Video: Una técnica ex vivo para generar células T receptoras de antígenos quiméricos específicos de tumores - Concepto

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

Un modelo con ratones Syngeneic células B linfoma de evaluación preclínica de las células de T automóvil CD19

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Investigación

JoVE Journal

Investigación sobre el cáncer

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Uso de CRISPR/Cas9 para noquear GM-CSF en células CAR-T

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

Transcripción

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells