iGEM 2025 — Team lzu-china

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{% block title %}Project Description{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
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Notebook

Design

Schedule

Results

Conclusion

Iteration Plan

Design

This section falls under the exosome delivery efficacy validation module within the overall project design.
The design encompasses miRNA-loaded exosome delivery methods, with the specific workflow and procedures as
follows:
The experimental section is mainly divided into three parts:construction of engineered exosomes,establishment
of and intervention with an in vitro hepatic fibrosis model,evaluation and validation of anti-fibrotic
effects.

Schedule

Overall experimental plan

| 2025.8.14 | 2025.8.25 | 2025.9.5 |
| --- | --- | --- |
| Exosomes and RNA Ordering | RNA transfection into EVs and Hepatic Stellate Cell Culture | miRNA Quantification and Functional Validation |
| --- | --- | --- |

Exosomes and RNA Ordering

Director：Nuo Xu,Boliang Liu
Begin at：2025.8.14
Estimated end date ：2025.8.24
Current completion status：finished（ 2025.8.24）

RNA transfection into EVs and Hepatic Stellate Cell Culture

**Part One: Hepatic Stellate Cell Culture**
Director：Nuo Xu,BoLiang Liu
Begin at：2025.8.15
Estimated end date ：2025.9.1
Current completion status： Finished（ 2025.9.16）
**Part Two: In Vitro Validation of Anti-fibrotic Effects**
Director：Nuo Xu，BoLiang Liu
Begin at：2025.9.18
Estimated end date ：2025.9.20
Current completion status ：Finished（ 2025.9.21）
Record of project:
Step 1: Establishment of a Hepatic Fibrosis Model
LX-2 cells were cultured in 24-well plates.
To induce cell activation and establish a hepatic fibrosis model, the cells were treated with TGF-β1 (10
ng/mL) for 24 hours.
Step 2: Experimental Group Setup
The following seven experimental groups were established for intervention:
1.Negative Control Group: Untreated normal LX-2 cells.
2.Positive Model Group: LX-2 cells induced by TGF-β1 only.
3.Natural Exosome Group: Treated with TGF-β1 and natural exosomes.
4.NC Exosome Group: Treated with TGF-β1 and exosomes loaded with NC mimic.
5.miR-455-3p Exosome Group (Group A): Treated with TGF-β1 and exosomes loaded with miR-455-3p mimics.
6.miR-148a-5p Exosome Group (Group B): Treated with TGF-β1 and exosomes loaded with miR-148a-5p
mimics.
7.Co-delivery Exosome Group (Group A+B): Treated with TGF-β1 and a mixture of exosomes from Group A and B,
at three different miRNA ratios (1:1, 1:2, 2:1).
Step 3: Exosome Intervention
Based on pre-determined actual miRNA content in each exosome preparation, the concentrations for different
groups were precisely calculated and prepared.
This ensured a constant total exosome delivery concentration (50 µg/mL) while maintaining accurate and
controllable miRNA ratios.

miRNA Quantification and Functional Validation

Director：Nuo Xu,Boliang Liu,Zewen Zhang
Begin at：2025.9.21
Estimated end date ：2025.9.30
Current completion status： Finished（ 2025.10.4） Record of project:
Step 1: Cell Collection
After the specified intervention time (e.g., 48h, 72h), cells from all groups were collected.
Step 2: Protein Extraction and Western Blot
Total protein was extracted from the collected cells.
The protein expression levels of α-SMA and Collagen I were detected by Western Blot analysis.
Step 3: Data Analysis
Grayscale analysis was performed on the Western Blot results.
The expression differences of α-SMA and Collagen I among the groups were compared to evaluate the
synergistic anti-fibrotic effect of the dual-miRNA co-delivery and to determine the optimal effective
molecular ratio.

Results

Results of miRNA transfection into exosomes

**kit method**
Based on the results of fluorescence detection, we can infer that miR-148a-5p has been almost
entirely transfected into the exosomes. A portion of the miRNAs in the miR-455-3p group were
transfected into the exosomes, thus obtaining exosomes loaded with these two
miRNAs.

Measuring the OD value to determine whether miRNA has been transferred into
exosomes. The fluorescence report is as follows:

miR-455-3p:

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/1-1.webp)

miR-148a-5p:

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/1-2.webp)

**co-incubation method**
The exosomes loaded with miRNA provided by the company are as follows

Exosomes loaded with miRNA:

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/1-3.webp)

cell culture

After being cultured in complete medium, resuscitated hepatic stellate cells (HSCs) exhibit the
characteristics of a significant increase in density and a morphological transition from the
quiescent state to the activated state under an optical microscope. At this stage, the cells have
passed the initial adaptation phase and entered the late logarithmic growth phase; in terms of
density, they can cover 70%-90% of the bottom area of the culture dish, with a slightly sparse
distribution at the edges and a uniform distribution in the central area. Morphologically, the cells
no longer have the round or oval shape (in the state of incomplete adherence or just adherent)
observed in the early resuscitation stage, but transform into a spindle-shaped, polygonal, or long
spindle-shaped morphology similar to myofibroblasts. Their cell bodies are significantly stretched
with increased volume and abundant cytoplasm; the cell processes increase in number and length, and
interconnect with the processes of adjacent cells to form a network structure.

Changes in Cell Density During Cell Culture (Taking Cell Resuscitation to the First Passage as an
Example)

Day1

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-1-new.webp)

Day2

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-2.webp)

Day3

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-3.webp)

Day4

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-4.webp)

Day5

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-5.webp)

Day6

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-6.webp)

Pathological state of induced cells

Before induction, quiescent hepatic stellate cells (HSCs) adhere to the substrate and grow. They
exhibit a typical "spindle-shaped" or "polygonal" morphology, with small cell bodies. Additionally,
the cells extend multiple long, slender cytoplasmic protrusions that interconnect to form a
network-like structure.After induction with TGF-β, cell spacing decreases, and cell stacking even
occurs in some regions. Meanwhile, the cell morphology transitions from "spindle-shaped" to
"fusiform" (similar to fibroblasts). The cell bodies increase significantly in size, and the
cytoplasmic staining deepens—this is due to increased expression of cytoskeletal proteins such as
α-smooth muscle actin (α-SMA) in the cytoplasm. Furthermore, the original long, slender protrusions
shorten and thicken, and the intercellular connections decrease.

LX-2 cells in normal condition

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-7.webp)

LX-2 cells induced for 24 hours

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-8.webp)

LX-2 cells induced for 48 hours

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-9.webp)

LX-2 cells induced for 72 hours

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-10.webp)

Conclusion

Both the ExoLoad® Exosome Nucleic Acid Loading Kit and co-incubation method successfully engineered exosomes.

Iteration Plan

Based on the above results, we will further carry out multi-metric validation experiments for more delivery
ratios. This includes testing multiple fibrosis markers at different time points after drug delivery.

![LZU Logo](https://static.igem.wiki/teams/5547/logo/huojian.webp)

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

Overview

Cycle 1

Cycle 2

Overview

In order to provide an experimental basis and theoretical foundation for the development of new strategies for
efficient and multi-targeted hepatic fibrosis treatment, we constructed and optimized an engineered exosome
based on the synergistic action of dual miRNAs (miR-455-3p and miR-148a-5p) and systematically evaluated its
interventional effect on hepatic fibrosis.

Specific sub-objectives include:
1. To successfully construct engineered human umbilical cord mesenchymal stem cell exosomes (hUC-MSC-Exos)
loaded with miR-455-3p and miR-148a-5p, respectively.
2. To clarify the inhibitory effects on the proliferation, activation and extracellular matrix secretion of
activated hepatic stellate cells (HSCs) when the two engineered exosomes were applied singly and in
combination in different ratios.
3. To validate and screen out the combination ratio of dual miRNA engineered exosomes with the best
anti-fibrotic effect in an in vitro model of liver fibrosis.

Cycle 1 Construction and Characterization of Engineered Exosomes

Design

To develop therapeutic strategies for end-stage liver disease, we designed an engineered exosome system that
co-delivers two anti-fibrotic miRNAs (miR-455-3p and miR-148a-5p) derived from human umbilical cord
mesenchymal stem cells (hUC-MSCs). The goal was to construct exosomes with enhanced miRNA loading capacity and
validate their physical and molecular characterization.

Build

We acquired hUC-MSCs (CTE-052) exosomes from ECHO AIOTECH. Synthetic miRNA mimics (miR-455-3p and miR-148a-5p)
were then loaded into exosomes using two methods: commercial loading kit, ExoLoad® and direct co-incubation.
Negative control (NC) mimics were also loaded for comparison.

Test

We used electron microscopy and nanoparticle tracking analysis (NTA) to characterize the morphology and size
distribution of exosomes. Exosome markers (e.g. CD9, CD63) were confirmed using protein blotting.
Quantification of miRNA content in engineered exosomes by fluorescence detection.

Learn

We successfully constructed engineered exosomes with high miRNA loading efficiency. By comparing the kit
method and the co-incubation method, we determined the best method for subsequent functional experiments.

Cycle 2: In vitro modeling of liver fibrosis and exosomal intervention

Design

To assess the anti-fibrotic effects of engineered exosomes, we designed an in vitro model of liver fibrosis
using a human liver stellate cell line (LX-2) activated by TGF-β1. The model mimics key pathological features
of liver fibrosis.

Build

LX-2 cells were cultured and treated with TGF-β1 (10 ng/mL) for 24 hours to induce their activation. The
cells were then divided into seven experimental groups:

| No. | Group name | Whether induced by TGF-β1 | Exosome type | Purpose |
| --- | --- | --- | --- | --- |
| 1 | Negative blank control | No | None | Observation of LX-2 cells in their natural state |
| 2 | Positive Blank Control | Yes | None | Observation of changes following LX-2 modelling at any manual intervention |
| 3 | Natural exosome control | Natural exosomes | Observation of the effect of natural |
| 4 | NC mimic loading exosome group | Ev transferred to rna | Exclusion of artificial RNA backbones from LX-2 cells |
| 5 | miR-455-3p exosome control (group A) | ev to 455 | Observation of the effect of miR-455-3p loaded exosomes on active LX-2 |
| 6 | miR-148a-5p exosome control group (Group B) | Transferred to 148 ev | Observation of the effect of miR-148a-5p loaded exosomes on active LX-2 |
| 7 | Experimental group ratio 1 (miR-455-3p :miR-148a-5p ) | 1:1 | Observation of the effect of ratio 1 engineered exosomes on active LX-2 |
| 8 | Experimental group ratio 2 | 2:1 | Observation of the effect of ratio 2 engineered exosomes on active LX-2 |
| 9 | Experimental group ratio 3 | 1:2 | Observation of the effect of ratio 3 engineered exosomes on active LX-2 |

Test

After intervention (e.g., 48 or 72 hours), cells were collected. Protein extracts are prepared and tested for
fibrosis markers such as α-SMA by protein blot analysis.

Learn

We found that engineered exosomes would significantly inhibit HSC activation and extracellular matrix
production. Preliminary data which we haven’t acquired can show whether single miRNA exosomes are (or are not)
superior to natural exosomes and whether co-delivery is (or is not) showing enhanced effects.

![LZU Logo](https://static.igem.wiki/teams/5547/logo/huojian.webp)

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Project Description{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
{% block page\_content %}

Results

Results of miRNA transfection into exosomes

cell culture

Pathological state of induced cells

Results of miRNA transfection into exosomes

kit method

Based on the results of fluorescence detection, we can infer that miR-148a-5p has been almost
entirely transfected into the exosomes. A portion of the miRNAs in the miR-455-3p group were
transfected into the exosomes, thus obtaining exosomes loaded with these two
miRNAs.

Measuring the OD value to determine whether miRNA has been transferred into
exosomes. The fluorescence report is as follows:

miR-455-3p:

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/1-1.webp)

miR-148a-5p:

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/1-2.webp)

co-incubation method

The exosomes loaded with miRNA provided by the company are as follows

Exosomes loaded with miRNA:

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/1-3.webp)

cell culture

After being cultured in complete medium, resuscitated hepatic stellate cells (HSCs) exhibit the
characteristics of a significant increase in density and a morphological transition from the
quiescent state to the activated state under an optical microscope. At this stage, the cells have
passed the initial adaptation phase and entered the late logarithmic growth phase; in terms of
density, they can cover 70%-90% of the bottom area of the culture dish, with a slightly sparse
distribution at the edges and a uniform distribution in the central area. Morphologically, the cells
no longer have the round or oval shape (in the state of incomplete adherence or just adherent)
observed in the early resuscitation stage, but transform into a spindle-shaped, polygonal, or long
spindle-shaped morphology similar to myofibroblasts. Their cell bodies are significantly stretched
with increased volume and abundant cytoplasm; the cell processes increase in number and length, and
interconnect with the processes of adjacent cells to form a network structure.

Changes in Cell Density During Cell Culture (Taking Cell Resuscitation to the First Passage as an
Example)

Day1

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-1-new.webp)

Day2

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-2.webp)

Day3

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-3.webp)

Day4

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-4.webp)

Day5

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-5.webp)

Day6

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-6.webp)

Pathological state of induced cells

Before induction, quiescent hepatic stellate cells (HSCs) adhere to the substrate and grow. They
exhibit a typical "spindle-shaped" or "polygonal" morphology, with small cell bodies. Additionally,
the cells extend multiple long, slender cytoplasmic protrusions that interconnect to form a
network-like structure.After induction with TGF-β, cell spacing decreases, and cell stacking even
occurs in some regions. Meanwhile, the cell morphology transitions from "spindle-shaped" to
"fusiform" (similar to fibroblasts). The cell bodies increase significantly in size, and the
cytoplasmic staining deepens—this is due to increased expression of cytoskeletal proteins such as
α-smooth muscle actin (α-SMA) in the cytoplasm. Furthermore, the original long, slender protrusions
shorten and thicken, and the intercellular connections decrease.

LX-2 cells in normal condition

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-7.webp)

LX-2 cells induced for 24 hours

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-8.webp)

LX-2 cells induced for 48 hours

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-9.webp)

LX-2 cells induced for 72 hours

![](https://static.igem.wiki/teams/5547/protocal-and-result-page/2-10.webp)

![LZU Logo](https://static.igem.wiki/teams/5547/logo/huojian.webp)

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