Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with >85% transduction.
Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce >85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.
Immunotherapy has emerged as a revolutionary cancer treatment paradigm due to its ability to specifically target tumors, limit off-target cytotoxicity, and prevent relapse. Particularly, chimeric antigen receptor T (CAR-T) cell therapy has gained popularity due to its success in treating lymphomas and leukemias. The FDA approved the first CAR-T cell therapy in 2017, and, since then, has approved four more CAR-T cell therapies1,2,3,4,5. CARs have an antigen recognition domain usually consisting of a single chain variable fragment of a monoclonal antibody that is specific for a tumor associated antigen3,4. When a CAR interacts with its tumor-associated antigen, the CAR-T cells become activated, leading to an antitumor response involving cytokine release, cytolytic degranulation, transcription factor expression, and T cell proliferation. To produce CAR-T cells, blood is collected from the patient to obtain their T cells. CARs are genetically added to the patient's T cells using a virus. The CAR-T cells are grown in vitro and infused back into the patient2,3,4,6. Successful generation of CAR-T cells is determined by the transduction efficiency, which describes the number of T cells that are genetically modified into CAR-T cells.
Currently, the gold standard for CAR-T cell generation is spinoculation of activated T cells and virus on retronectin-coated plates7,8. Transduction begins when viral particles engage with the surface of the T cells. Retronectin promotes colocalization of virus and cells by increasing the binding efficiency between the viral particles and the cells, enhancing transduction7,8. Retronectin does not work well on its own and needs to be accompanied by spinoculation, which enhances gene transfer by concentrating the viral particles and increasing the surface permeability of the T cell, allowing for easier viral infection8. Despite the success of spinoculation on retronectin-coated plates, it is a complex process that requires multiple spin cycles and expensive reagents. Therefore, alternate methods for viral gene transfer that are quicker and cheaper are highly desirable.
Alginate is a natural anionic polysaccharide extensively used in the biomedical industry due to its low cost, good safety profile, and ability to form hydrogels upon mixing with divalent cations9,10,11,12. Alginate is a GMP-compliant polymer and is generally recognized as safe (GRAS) by the FDA13. Cross-linking alginate with cations creates stable hydrogels often used in wound healing, delivery of small chemical drugs and proteins, and cell transportation9,10,11,12,14,15,16. Due to its excellent gelling properties, alginate is the preferred material to create porous scaffolds by freeze-drying10,17. These characteristics of alginate make it an attractive candidate for producing a scaffold that can mediate viral gene transfer of activated cells.
Described here is a protocol for making dry macroporous alginate scaffolds, known as Drydux scaffolds, that statically transduce T cells by viral gene transfer17,18. The process for making these scaffolds is shown in Figure 1. These scaffolds eliminate the need for spinoculation of retronectin-coated plates. The macroporous alginate scaffolds encourage the interaction of viral particles and T cells to enable efficient gene transfer in a single step without affecting functionality and viability of the engineered T cells17. When followed correctly, these macroporous alginate scaffolds have a transduction efficiency of at least 80%, simplifying and shortening the viral transduction process.
Figure 1: Schematic and timeline of the protocol. (A) Timeline for making the dry macroporous alginate scaffolds. Alginate is cross-linked with calcium-D-gluconate and frozen overnight. The frozen scaffolds are lyophilized for 72 h to create the Drydux scaffolds. (B) Timeline for viral transduction of activated cells. Activated cells and virus (MOI 2) are seeded on top of the scaffold and incubated in complete media supplemented with IL-7 and IL-15. The scaffolds absorb the mixture and promote viral gene transfer. EDTA is used to dissolve the scaffolds and isolate the transduced cells. After washing twice with PBS, the cell pellet can be used for analysis. Abbreviations: PBS = phosphate-buffered saline; PBMCs = peripheral blood mononuclear cells. Please click here to view a larger version of this figure.
All the procedures involving human primacy cells and retroviral vectors were performed in compliance with North Carolina State University's Biological Safety guidelines and approved by the Environmental Health and Safety Office. Human peripheral blood mononuclear cells were purchased as buffy coats from commercial sources. Primary human cells must be isolated from human buffy coat fractions and require Biosafety Level 2 clearance and detailed standard operating procedures and approval from the institution where the work is to take place. Viral vectors, including the retroviral vector supernatants used for the transduction prepared as previously described19, can be classified either as Biosafety Level 1 or Biosafety Level 2 depending on the encoded protein and require approval from the relevant institutional biosafety committee.
1. Making the macroporous alginate scaffolds
2. Transduction
These macroporous alginate scaffolds are easy to make and should come out of the lyophilizer as porous, fluffy, and white discs. Although not studied in this experiment, calcium-alginate solution can be cast into different molds to create scaffolds of varying shapes, depending on the needs of the user9,10. The scaffolds are electrostatic and may stick to the lid of the well-plate or to a gloved finger. Figure 2 demonstrates what the scaffolds should look like upon completion. Approximate dimensions of 24- and 48-well scaffolds are also shown in the figure.
Figure 2: Images of dry macroporous alginate scaffolds. (A) A 48-well plate full of scaffolds. (B) A 24-well plate full of scaffolds. (C) Comparing a 48-well scaffold to a 24-well scaffold. Scaffold dimensions are also shown. Please click here to view a larger version of this figure.
The porosity of the scaffolds is highly important for successful transduction, and we have previously experimented with the porosity of the scaffolds. For more information regarding the porosity, including SEM images of the scaffolds, please refer to papers by Agarwalla et. al. in the references17,18.
Transduction efficiency was analyzed using flow cytometry and the results are shown in Figure 3. Frozen PBMCs from the same donor were activated and used for all experimental groups. A protocol for activating PBMCs can be found in Supplemental File 1, and any standard method to activate T cells and validate activation can be used21,22,23. The activated PBMCs were seeded on either a scaffold with GFP-encoding retrovirus or a scaffold without virus, termed a "blank" scaffold. Activated PBMCs were also seeded on plates spinoculated with retronectin and GFP-encoding retrovirus to compare the transduction efficiency of the alginate scaffolds to the conventional method. Non-transduced (NT) cells-activated PBMCs seeded in uncoated wells of a 24-well plate-were used as the negative control group. As expected, the non-transduced cells and cells isolated from the blank scaffold did not show any transduction. Cells isolated from the scaffold seeded with activated PBMCs and GFP retrovirus showed comparable transduction efficiency to the retronectin-coated plates. The scaffold had an average transduction efficiency of 85%, just below the retronectin group. These results demonstrate that these alginate scaffolds serve as an easier and cheaper alternative to virally transduce T cells without the need for spinoculation of retronectin.
Figure 3: FACS quantification of transduction efficiency. (A) FACS plots showing GFP expression. Cells were gated on viable cells, FSC singlets, and GFP positive cells. (B) Quantification of GFP-positive cells by FACS. Conventional spinoculation of retronectin-coated plates (purple inverted triangle) was used as a positive control. Non-transduced cells (blue circle) and activated cells seeded on the alginate scaffolds without virus (red square) were used as negative controls. Scaffolds seeded with activated cells and GFP retrovirus (green triangle) had a transduction efficiency of 85%, comparable to retronectin. Data represented as mean ± standard deviation with n = 4. Abbreviations: GFP = green fluorescent protein; FACS = fluorescence-activated cell sorting; SSC-A = side scatter-peak area; FSC = forward scatter. Please click here to view a larger version of this figure.
Supplemental File 1: Protocol for activating cells. Please click here to download this File.
Supplemental Figure S1: Images of dry macroporous alginate scaffolds fabricated at -80 ˚C. Freezing scaffolds at -80 ˚C leads to less consistent scaffold appearance and function than when frozen at -20 ˚C. Please click here to download this File.
CAR-T cell therapy continues to gain interest for both research and commercial applications. Despite the success CAR-T cell therapy has had in treating blood cancers, the high cost of the procedure limits its use. The protocol presented here introduces a new method for viral gene transfer of T cells without the need for spinoculation of retronectin-coated plates. Producing dry macroporous alginate scaffolds to mediate transduction is relatively simple and is a suitable low-cost replacement for the conventional method.
Occasionally, scaffold appearance may differ from those shown in Figure 2 and may instead appear clear and crystalline rather than white and fluffy. This can occur from inconsistent freezing at -20 ˚C due to repeated opening of the freezer door or if the plate to be frozen is insulated by being next to or on top of bulky items in the freezer. Despite the difference in appearance, our lab has not experienced any difference in transduction efficiency with the crystalline scaffolds. Although previous work on cryogelation has used lower freezing temperatures24,25, we found that freezing at lower temperatures leads to less consistent scaffold appearance and function than when frozen at -20 ˚C. Images of a scaffold created by freezing at -80 ˚C can be found in Supplemental Figure S1. Therefore, it is still recommended that scaffolds should be frozen at -20 ˚C with minimal disruption, and the plate should be placed on a rack-style shelf that allows air flow under the plate.
Although these scaffolds have shown excellent transduction efficiency, a few limitations should be noted. First, care must be taken when working with EDTA, which can limit cell viability, even in small amounts. Hence, EDTA should be completely removed. Additionally, scaffold size will limit the amount of liquid that can be absorbed and, thus, the number of cells that can be transduced. Finally, this protocol cannot be used to transduce unactivated T cells. Future work will focus on reporting fabrication of scaffolds capable of simultaneously mediated activation and proliferation of cells, promoting colocalization of cells and viral particles for transduction, and releasing fully functional transduced cells in vivo18.
Published studies by our laboratory report the phenotypic analysis and functionality of CAR-T cells generated using these scaffolds. The CD19-specific CAR-T cells produced by these scaffolds retained effector phenotype and were able to eliminate CD19+ Daudi cells in vitro when co-cultured at a 1:5 effector-to-target ratio17. The CAR-T cells co-cultured with Daudi cells also released IL-2 and IFN-γ, which are proinflammatory cytokines that indicate that the T cells are activated. These results demonstrate that these scaffolds produce highly functional CAR-T cells in vitro. CAR-T cells generated by these scaffolds showed excellent in vivo antitumor function against CD19+ Daudi cells labeled with firefly luciferase. The CAR-T cells effectively controlled tumor growth, improved overall survival rate, and did not show any significant signs of toxicity17. These results indicate that these scaffolds can be used to genetically modify cells for CAR-T cell therapy without affecting the functionality and antitumor activity of the cells. Future studies with these alginate scaffolds involve optimizing transduction efficiency by adjusting the macroporosity as well as alginate and calcium concentrations.
In conclusion, these macroporous alginate scaffolds have a comparable transduction efficiency to retronectin, providing an alternative method for transducing cells for CAR-T cell therapy. These scaffolds also produce fully functional CAR-T cells with antitumor activity both in vitro and in vivo17. Drydux scaffolds offer an alternative method that is simple and inexpensive for transducing T cells for use in CAR-T cell therapies.
The authors have nothing to disclose.
This work was supported by the National Institutes of Health through Grant Award Numbers R37-CA260223, R21CA246414. We thank the NCSU flow cytometry core for training and guidance on flow cytometry analysis. Schematics were created with Biorender.com
0.5 M EDTA | Invitrogen | 15575-038 | UltraPure, pH 8.0 |
1x DPBS | Gibco | 14190-144 | No calcium chloride or magnesium chloride |
3% Acetic Acid with Methylene Blue | Stemcell Technologies Inc | 07060 | |
Activated Periphreal Blood Mononuclear Cells | – | – | Fresh or frozen |
Calcium-D-Gluconate | Alfa Aesar | A11649 | |
CD28.2 Antibody | BD | 555725 | 1 mg/mL |
CD3 Antibody | Miltenyi | 130-093-387 | 100 μg/mL |
Click's Media | FUJIFILM IRVINE SCIENTIFIC MS | 9195 | |
DI Water | – | – | |
Glutamax | Gibco | 35-050-061 | |
HyClone FBS | Cytvia | SH3039603 | |
HyClone RPMI 1640 Media | Cytvia | SH3009601 | |
Penicillin-streptomycin (P/S) | Gibco | 15-140-122 | |
Peripheral Blood Mononuclear Cells | – | – | Fresh or frozen |
PRONOVA UP MVG | NovaMatrix | 4200101 | Sodium alginate |
Recombinant Human IL-15 | Peprotech | 200-15 | 5 ng/mL |
Recombinant Human IL-7 | Peprotech | 200-07 | 10 ng/mL |
Retrovirus | – | – | 1 x 106 TU/mL |