Engineering

"Some of the best lessons we ever learn are from our mistakes and failures. The error of the past is the wisdom and success of the future." - Tryon Edwards

We employed engineering principles in the creation and construction of components within our biological system. Furthermore, we adopted the engineering design cycle to address the various challenges in our project. This design cycle comprises four key stages: Design, Build, Testing, and Learning. These cycles show how we tackled challenges during our project, what we learned from each engineering iteration, and what we did to improve the design.

We also integrated the aspect of Research, as it is frequently crucial for the effective design of a system. The engineering design cycle is visualized in Figure 1.

On this engineering success wiki page, we discuss each step of our experimental workflow from the NK expansion and activation to transduction according to the DBTL cycle. Each step is accompanied by an elaborated explanation, including results, thoughts, and decisions.

1.Activating Natural Killer Cells

Since the first arrival iPSCs- derived NK cell kit, led to unsuccessful culturing and expansion trials of these cells, we aimed to utilize the time frame between a new kit delivery, in order to evaluate the capacity of different conditions in activating Natural Killer cells. For this purpose, we utilized Natural Killer cells isolated from the Peripheral Blood.

Design:

We planned to test three different activating conditions, maintaining a standard cytokine mix (IL-2, IL-15, IL-21). Baseline condition contained only cytokines. In addition to the cytokine mix, the next condition contained the Cloudz NK activation kit, enriched with anti-CD2 and anti-NKp46 antibodies. For the Feeder-Cells condition, irradiated K562 cells, treated with mitomycin were co-cultured with NK cells, also in addition to cytokine mix.

Build:

Activation capacity, of each setted condition, was evaluated with FACS analysis. Cells were stained with anti- CD56 antibody, a well- established surface marker of NK cells, which quantifies the sample, along with an CD25 antibody, an activation marker.

Test:

The result was that NK cells activate best in the presence of feeder cells (43.6 % of NK cells were activated compared to 22.5% for baseline and 14.3% for Cloudz activated).

Learning

FACS analysis revealed strong activation of NK cells, in presence of irradiated and mitomycin treated K562 feeder cells, compared to cloudz and baseline conditions. So We decided to utilize K562 feeder cells for our NK cell cultures.

2.Transfection experiment

Design

HEK 293 cells were co-transfected with three 2nd generation lentiviral plasmids for lentiviral particle formation. These plasmids carry the lentiviral genome, which has been modified for safety reasons, retaining only the genes necessary for lentiviral vector synthesis and thereby preventing any replication capability. In the 2nd generation lentiviral plasmid system, the viral genetic material is fragmented into three separate plasmids.

Build

Transfection Protocol includes the detailed methodology, from preparing HEK-293 cell line, to transfection finalized with Flow Cytometry aiming to detect eGFP, contained in transfer plasmid, which carries our Chimeric Antigen Receptor.

The three separate plasmids for the lentiviral particle formation are the following:

Envelope Plasmid: pMD.2G

Packaging Plasmid: psPAX2

Transfer Plasmid: pGenLenti-IRES-eGFP-BleoR

Testing

The pGenlenti-GFP plasmid successfully transfected into HEK cells and demonstrated GFP expression.

Learning

The successful expression of GFP in HEK cells is an indication that the plasmid has some functionality and the IRES element works. The low transfection rate challenges us to find better transfection conditions.

3.Transduction experiment

Design

Induced Pluripotent Stem Cells (iPSCs)-derived NK cells and NK-92 cell line, were transduced with lentiviral vectors, produced previously, aiming to express our anti-mesothelin-CAR.

Build

We concentrated the viral supernatant that HEK-293 produced and we transduced both (iPSCs)-derived NK cells and NK-92 cell line.

Testing

In the flow cytometry analysis , we did not get a GFP positive sample.

Learning

After analyzing the experimental steps that were followed, attention was directed towards the possible issues that led to the absence of GFP expression after virus packaging. So we concluded that the virus packaging process is likely responsible. It is possible that there were insertion or integration issues with the plasmid during the packaging process preventing the expression of the GFP gene. As next steps we could try to verify if the plasmid was correctly inserted into the viral vector or confirm if the plasmid was integrated into the cell genome. It is also possible that the amount of viral particles was not enough to transduce the cells, so we could try to produce more viral particles by using a bigger quantity of plasmid at transfection.

Future steps

Our future goals as Wet Lab are to achieve the viral transduction of iPSCs derived NK cells in order to test the cytotoxicity the CAR-NKs in the AsPC-1 pancreatic cancer cell line, which we have seen from the literature that overexpresses mesothelin. This cytotoxicity experiment will prove whether the chimeric antigen receptor we designed is functional. Furthermore, we will focus on GMP (Good Manufacturing Practice) culture conditions, so that the final cellular product resulting from this process is as safe as possible for the patient. Also, we could try different protocols for the transfection and use lipofectamine, a reagent commonly used for such processes. Different transduction techniques, like electroporation could be utilized also. We could also build a 3D printed bioreactor to produce our cells in large quantities and under controlled conditions.