iGEM 2023 — Team patras-med

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

### 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.

![](https://static.igem.wiki/teams/5017/wiki/engineering.png)

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

**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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) , 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.

1. [Engineering](#Engineering)
2. [1.Activating Natural Killer Cells](#1.Activating Natural Killer Cells)- [2.Transfection experiment](#2.Transfection experiment)
   - [3.Transduction experiment](#3.Transduction experiment)
   - [Future steps](#Future steps)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5017/wiki/experiments/experiments.png)

### Antigen Selection Strategy

Antigen selection was initially performed, utilizing existing available literature, focused on the analysis of
the
histopathology of pancreatic cancer and evaluating expression levels of certain antigens in the cancerous
tissue,
compared to normal tissue. In the meantime, the role of each overexpressed membrane bound antigens had to be
clarified
to find its association with cancer prognosis and thus detect a highly specific antigen. In this manner, an
initial
pool of specific antigens was chosen which subsequently underwent further investigation through utilization of
the
**Geo2R analysis tool.**

![](https://static.igem.wiki/teams/5017/wiki/experiments/pinakas-ex.png)

The Gene Expression Omnibus (GEO) serves as a repository database that houses a significant portion of
publicly
accessible high-throughput gene expression data. The analysis of gene expression represents a potent
instrument for
acquiring understanding regarding the mechanisms and procedures that underlie the biological and phenotypic
distinctions observed among various sample groups. By leveraging the data within GEO2R, we can identify
differentially
expressed genes in pancreatic cancer tissues compared to normal tissue, providing insights into potential
target
candidates. This strategy allows for the systematic evaluation of gene expression profiles, enabling the
identification of specific antigens that play pivotal roles in the progression of pancreatic cancer.

LogFC stands for "log Fold Change". It is a statistical metric employed in the examination of gene expression
data,
primarily when comparing gene expression levels across different sets of groups or conditions. Specifically,
Fold
Change (FC) is quantifying the degree of alteration in a gene's expression level between two experimental
conditions.
This metric helps researchers identify genes that are significantly upregulated (positive logFC) or
downregulated
(negative logFC) between the conditions being compared. It is a crucial indicator for understanding which
genes are
differentially expressed and to what extent in a given study. **Positive logFC:** indicates a
significant
upregulation of
a gene in the treatment group compared to the control. **Negative logFC:** indicates a
significant
downregulation. A LogFC
value in close proximity to zero signifies minimal to negligible fluctuations in gene expression between the
two
sample groups. Genes characterized by logFC values near zero are regarded as exhibiting similar expression
levels in
both groups.

Through extensive analysis of gene expression data within the database, we have consistently observed
overexpression
of mesothelin in pancreatic cancer tissues when compared to normal tissues. This stark contrast makes
mesothelin an
attractive candidate for targeted therapies, as it is an antigen that is not only abundant in cancer cells but
also
relatively absent in healthy tissues. The utilization of GEO2R has played a pivotal role in the discovery and
validation of mesothelin as a target antigen, providing valuable insights into its potential as a therapeutic
target
for pancreatic cancer. From the volcano plot below we can clearly see that mesothelin is**overexpressed** in pancreatic
cancer and this overexpression is **statistically significant**, making mesothelin the**ideal
candidate antigen.**

![](https://static.igem.wiki/teams/5017/wiki/experiments/volcano-plot.png)

One more element that led us to the choice of mesothelin is that the protein has been crystallized and several
PDB
files have been deposited in the Protein Data Bank database. This provides our Dry-Lab with the ability to
perform
accurate molecular simulations and docking experiments.

### Plasmid Design Strategy

Proper design of our Chimeric Antigen Receptor (scFv-NKG2D-2B4-CD3z) is an essential step to prove our idea,
target
and kill cancer cells in pancreatic cancer. Because there was no experience in previous plasmid design we
ended up
with this proposed plasmid design strategy: the sequence of the desired genes that make up the receptor were
found
from the National Center for Biotechnology Information (NCBI) database. However, in the transfer plasmid, only
a
section of the complete sequence should be integrated, necessitating the identification of this specific
segment. For
this purpose, we utilized existing plasmids that are deposited in the Addgene repository and consist of our
structural
units. Then, the exact sequence of these genes that have been used in the deposited plasmids, were searched
using the
BLAST global alignment tool. By this method we were led in a more secure way to the total sequence of the
plasmid.
Below you can see the use of this strategy for the identification of the CD3z domain.

![](https://static.igem.wiki/teams/5017/wiki/experiments/cd3zeta.png)

![](https://static.igem.wiki/teams/5017/wiki/experiments/addgene.png)

![](https://static.igem.wiki/teams/5017/wiki/experiments/next.png)

![](https://static.igem.wiki/teams/5017/wiki/experiments/2.png)

### Culturing cells

Care and maintenance of cell health ensures their functionality, expansion, proper preparation for
transduction
and
their cytotoxic efficacy. Corresponding protocols include initial thawing
process, culturing and feeding.
Specifically, it is detailing the thawing procedure, a critical step that significantly influences the
subsequent
progression of the culture, as it can lead to a potential reduction of up to 50% in the cell count.
Additionally, the
protocols provide instructions regarding how often to replenish nutrients, the quantity to administer, and the
method
for cell counting subsequent to staining with Trypan Blue. Cancer cell line AsPC-1 is also included in
protocols
concerning mammalian cell lines.

### Testing Conditions

After receiving cells, we had the time to test different activation conditions for Natural Killer Cells
obtained
from
a Peripheral Blood sample, provided by the laboratory we worked in. These Condition Testing Protocols were
created
based on bibliography research and include PBMCs isolation, Natural Killer cells selection, three different
culturing
conditions, further tested with Flow Cytometry. In these three conditions, we maintained a standard cytokine
cocktail
of IL-2, IL-25 and IL-21, adding the anti-CD2 and anti-NKp46 antibodies in cloudz condition, while the third
one
contained K562 feeder cells previously treated with mitomycin C and irradiation. In this way, we could detect
differences in NK activation efficacy and finally use the best condition as an alternative culture medium for
iPSCs-NK
activation.

### Preparing for Transfection

Plasmid is delivered in a lyophilized form and has to be multiplied in order to get sufficient final quantity.
For
this purpose we utilized the E. coli DH5α for initial plasmid transformation, followed by plasmid mini prep
and
mega
prep, aiming to harvest high plasmid concentrations before transfecting HEK 293 cells. Preparation for transfection
protocols include in detail experiments concerning culturing E. coli (DH5α) in agar, plasmid transformation,
colony
selection, mini prep and mega prep.

### Transfection

HEK 293 cells were co-transfected with three plasmids for lentiviral particle formation. Transfection Protocol
includes the detailed methodology, from preparing HEK-293 cell line, to transfection and supervision of the
progress,
finalized with Flow Cytometry aiming to detect eGFP, contained in transfer plasmid, transferring gene encoding
our
Chimeric Antigen Receptor.

Envelope Plasmid: pMD.2G

Packaging Plasmid: psPAX2

Transfer Plasmid: pLenti-IRES-eGFP-BleoR

### Transduction

Induced Pluripotent Stem Cells (iPSCs)-derived NK cells and NK92 cell line, were transduced with lentiviral
vectors,
produced previously, aiming to the expression of our anti-mesothelin-CAR. Transduction Protocol includes
previously
counting of cells after staining with Trypan Blue for ensuring their health.

![](https://static.igem.wiki/teams/5017/wiki/experiments/wet-lab-timeline.png)

DAY 0 (11/08/2023)

Delivery of iPSCs-NK

Due to delays in the delivery of the package and an insufficient amount of dry ice, the first package of
cells
came
without dry ice and thus the cells were under stress. IPSCs-derived NK cells were promptly frozen at -80 C,
while we
proceeded to communicate for their replacement by sending a new vial.

DAY 1 (08/09/2023)

IPSCs- Derived NK cells first thawing

Feeding day. IPSCs-NK cells suffered from transport and thawing and their number decreased, thus we were not
able to
proceed with their activation. As we were waiting for the new vial to be delivered, we started planning an
optimal
activation process and culture conditions.

DAY 3 (15/09/2023)

Peripheral Blood Mononuclear Cell (PBMC) Isolation - Natural Killer Cells Selection

While waiting for the arrival of new iPSCs- NK vial, we decided to assess three different activation
conditions,
identifying the optimal one to be later applied in iPSCs-NK cells. For this purpose, the laboratory we
worked in
provided us with a blood sample and thus we proceed to isolate NK cells from PBMC.

DAY 4 (17/09/2023)

Feeding PBMC-NK

Feeding Day.

DAY 5 (19/09/2023)

Flow Cytometry

To evaluate the activation of NK cells, in each condition designed we proceed to Flow Cytometry Analysis.

DAY 6 (25/09/2023)

Plasmid prep

Our plasmid arrived, we diluted it and we transfected DH5α competent cells.

DAY 7 (26/07/2023)

Colony selection

After transformation, we proceeded to colony selection based on ampicillin resistance.

DAY 8 (27/09/2023)

Mini Prep

We continued with mini prep, for an initial isolation of our DNA plasmid and evaluation of its
concentration.

DAY 9 (28/09/23)

Mega Prep

Mega Prep followed aiming to higher plasmid yield for proper preparation of transfection.

DAY 10 (29/09/23)

AsPC-1 thawing.

DAY 11 (02/10/23)

iPSCs-NK thawing, HEK 293 split and seed, K562 preparation

We proceeded to thaw the newly delivered iPSCs-NK vial, while preparing for HEK 293 transfection, that was
provided by
the laboratory we worked in. Thus, HEK 293 cells underwent split and seed. We also proceed to re-preparation
of
K562
feeder cells, by adding mitomycin and irradiating them.

DAY 12 (03/10/23)

Transfection

We immediately proceed to transfection of the HEK 293 cell line.

DAY 13 (05/10/23)

NK-92 thawing

This cell line was supplied to us by Mrs. Leukothea Papadopoulou's laboratory in order to have a cell line
easily
transduced, as an alternative. Delays occurred in its delivery, as it coincided with the days when floods
broke
out in
Thessaly, which led to the closure of roads and transport for almost two weeks.

DAY 14 (06/10/23)

Harvesting Virus - Transduction - Flow Cytometry

We gathered the virus produced so far and proceeded with the transduction of both iPSC-derived NK cells and
the
NK-92
cell line. This resulted in two sets of conditions for each, one that was transduced and the other that
remained
untransduced. Flow Cytometry for eGFP expression in HEK293, was also carried out, proving successful
transfection.

DAY 15 (07/10/23)

Change Media

DAY 16 (09/10/23)

Flow Cytometry

We evaluated the transduction efficacy. We tested the expression of eGFP, in both iPSC-derived NK cells and
the
NK-92 cell line.

Click here to see our lab book.

[lab-notebook.pdf](https://static.igem.wiki/teams/5017/wiki/experiments/lab-notebook.pdf)

1. [Antigen Selection Strategy](#Antigen Selection Strategy)
2. [Plasmid Design Strategy](#Plasmid Design Strategy)
3. [Culturing cells](#Culturing cells)
4. [Testing Conditions](#Testing Conditions)
5. [Preparing for Transfection](#Preparing for Transfection)
6. [Transfection](#Transfection)
7. [Transduction](#Transduction)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

### Result 1: NK Expansion & Activation Experiment

![](https://static.igem.wiki/teams/5017/wiki/photos-of-lab/pbmcs-nk.png)

In this particular experiment, three different conditions of NK cell activation were tested. Baseline had only the
cytokine mix present. For the Cloudz, the Cloudz NK activation kit was used in addition to cytokine mix. For the
Feeder-Cells condition, irradiated K562 cells were co-cultured with NK cells, also in addition to cytokine mix. Cells
were stained with an antibody to CD56, a classical surface marker of NK cells and with an antibody to CD25 antigen,
which is a marker of their activation. The result we got from this experiment is that NK cells activate best in the
presence of feeder cells: Feeder-Cells>Baseline>Cloudz, so we decided to utilize K562 cells for our cell cultures.

### Result 2: Transfection Experiment

![](https://static.igem.wiki/teams/5017/wiki/photos-of-lab/hek.png)

Singlets (94%): This gate represents the percentage of cells that are considered single cells, meaning they are not
clumped together or in aggregates. The fact that 94% of the cells fall into this category indicates that the majority
of the cells analyzed are in a single-cell state, which is important for accurate flow cytometry measurements.

GFP Positive (18.1%): This gate represents the percentage of cells that express the green fluorescent protein (GFP),
which is encoded by the transfected vector. These cells are considered GFP positive because they have successfully
taken up the plasmid and expressed the GFP gene.

In summary, the flow cytometry analysis of HEK-293T cells transfected with the GFP vector indicates that the majority
of the cells are in a single-cell state (94%), allowing for reliable individual cell analysis. Among these singlet
cells, approximately 18.1% are GFP positive, demonstrating successful transfection and expression of the GFP gene.
This information is essential for us because it proves that **the plasmid we designed has some
functionality** and **the
IRES element does its work**. It also proves that **our transfection protocol is working**
although the transfection
efficiency is not very big. The relatively low transfection efficiency may be due to the small concentration of
plasmid used, as well as the transfection reagent. In future experiments we will modify the protocol using more
plasmid and different transfection reagents to see which works best.

### Result 3: Transduction Experiment

![](https://static.igem.wiki/teams/5017/wiki/photos-of-lab/ipscs-nk.png)

![](https://static.igem.wiki/teams/5017/wiki/photos-of-lab/nk-92-trans.png)

In a flow cytometry experiment aimed at assessing the transduction efficiency in iPSCs-NK, and NK-92 cells, it was
found that the transduction was not successful. The flow cytometry results revealed the absence of GFP expression,
indicating that the transduction process did not yield the desired outcome. This outcome suggests a need for further
optimization of the transduction protocol or exploration of alternative methods to achieve the introduction and
expression of the CAR-GFP synthetic gene in these cell types. A possible fact that led to this result is that the
transfection efficiency was relatively low and thus not enough viral particles were produced. Furthermore, considering
that the percentage of GFP-positive in transduced cells is almost zero, it is very possible that the virus could not
be packaged well during the transfection of HEK-cells.

1. [Result 1: NK Expansion & Activation Experiment](#Result 1: NK Expansion & Activation Experiment)
2. [Result 2: Transfection Experiment](#Result 2: Transfection Experiment)
3. [Result 3: Transduction Experiment](#Result 3: Transduction Experiment)

{% endblock %}