Week 1: Proyect

HTBAA Homework #1

(1) What you would like to biomanufacture and why?

• Describe the target product or output you would like to make.
• Why are you interested in making it? Your motivation might be utilitarian—for example, addressing a problem in health, sustainability, food, energy, or manufacturing—but it does not need to be. Your project might instead have a social, cultural, artistic, or personal motivation.
• Clearly articulate what you want to make, who or what it is for, and why you think it is interesting or important.

I would like to develop a platform for producing patient-derived intestinal organoids in a defined, reproducible, and cost-effective synthetic extracellular matrix (ECM). The long-term objective is to use this platform to study inflammatory bowel diseases (IBD), particularly Crohn’s disease and ulcerative colitis, and eventually generate patient-specific functional profiles and treatment-response predictions.

This project is directly connected to my current PhD research. I am working on intestinal biology and organoids, and I see the development of an IBD-oriented platform as a potential direction for the second stage of my doctoral research. The project would therefore allow me to combine my current work with synthetic biology, tissue engineering, automation, and computational analysis.

IBD remains a major challenge because its underlying mechanisms are complex and heterogeneous, and patients with the same diagnosis can exhibit different biological phenotypes and responses to treatment. Although organoids provide a useful model of human intestinal tissue, their broader application is limited by factors such as the complexity and variability of extracellular matrices, the need for multiple growth factors and specialized culture media, cost, and difficulties in standardizing and scaling the system.

For this reason, I am particularly interested in developing a platform that addresses not only the biological model but also its manufacturing requirements. The goal would be to develop a synthetic or semi-synthetic ECM and, where economically and technically feasible, lower-cost sources of the media components and growth factors required for intestinal organoid culture. This could make the system more reproducible, accessible, and compatible with automated and high-throughput applications.

The immediate output would be standardized intestinal organoids derived from individual patients. These organoids could be exposed to controlled inflammatory conditions or therapeutic compounds and analyzed through imaging and molecular profiling. In the longer term, the resulting data could be used to identify disease-associated phenotypes and develop predictive models of therapeutic response.

Thus, the project is intended initially as a research and translational platform for IBD, rather than as a direct replacement for conventional clinical diagnosis. Its broader objective is to create a scalable system in which patient-derived cells, synthetic biomaterials, biological signals, automation, and computational analysis can be integrated to study intestinal disease and, eventually, support more personalized approaches to treatment.

(2) What living system would you use?

• Propose at least one living-system manufacturing chassis that could produce your target—for example, a microbial, mammalian, or cell-free system.
• At a high level, describe how the system would make your target product. What goes into the system? What biological process or processes occur? What comes out?
• You do not need to design the complete biological system yet. Make reasonable assumptions where necessary and identify them.

The product I would initially like to biomanufacture is a synthetic or semi-synthetic intestinal ECM capable of supporting the growth and organization of patient-derived intestinal organoids.

Rather than trying to reproduce the entire natural intestinal ECM, I would aim to identify and reproduce the key physical and biochemical properties required for intestinal stem-cell maintenance, proliferation, differentiation, and three-dimensional organization.

A defined matrix could potentially provide:

In addition, I would investigate whether some of the growth factors required for organoid culture, such as Wnt, R-spondin, Noggin, and EGF, could be produced using cell-free protein synthesis.

These components could eventually form a modular culture system in which both the physical environment and the biological signals are more precisely controlled.

(3) How would you use automation?

• Identify at least one automation technology that you would combine with your living system.
• This could include a large-scale Cloud Lab, a desktop liquid-handling robot such as an Opentrons, a programmable microfluidics platform such as Nuclera’s, or another automation technology.
• Describe what part of the biomanufacturing workflow you would automate and why.

According to the plan we have been developing, automation would not be an accessory element, but a central component for making organoid culture reproducible, scalable, and eventually useful for comparing multiple patients and experimental conditions.

The first technology I would use would be a benchtop liquid-handling robot, such as Opentrons, integrated with 96- or 384-well plates.

In the first stage, I would automate:

• Preparation and dispensing of the synthetic ECM
• Addition of biopsy-derived cells
• Addition of media and growth factors
• Periodic media changes
• Treatment administration
• Experimental perturbations
• Sampling
• Plate preparation

This would reduce variability introduced by manual handling and standardize culture conditions across patients and experiments.

Automation workflow

A second level of automation would involve controlling culture conditions and experimental perturbations. Once the organoids are established, the robot could programmatically administer different concentrations of growth factors, signaling pathway modulators, inflammatory stimuli, or drug treatments.

This would make it possible to perform dose-response experiments and systematically compare how organoids derived from different patients respond.

In the context of IBD, this step would be particularly important because the goal would not simply be to culture organoids, but to generate an individual functional profile for each patient.

Automated phenotyping

The next step would be to incorporate automated microscopy and imaging.

An imaging system could automatically record:

• Organoid size
• Organoid number
• Morphology
• Structural organization
• Viability
• Other quantifiable phenotypes

These data could then be analyzed using computer vision or machine learning to detect differences that may not be easily identified through manual observation.

In this way, the system would move beyond simply automating culture and would also automate phenotypic measurement.

Automated molecular analysis

A subsequent step would be to automate sampling and molecular analysis.

Depending on the scale, the collection of cells or culture supernatants and the preparation of samples for transcriptomics, proteomics, or other analyses could be automated.

At a later stage, these data could be integrated with imaging data and experimental conditions to build computational models capable of identifying patterns associated with different IBD phenotypes or treatment responses.

The complete workflow

The complete workflow can therefore be summarized as:

Patient biopsy
      ↓
Isolation of intestinal cells
      ↓
Synthetic ECM
      ↓
Automated organoid culture
      ↓
Growth factors / media
      ↓
Inflammatory conditions or treatments
      ↓
Automated imaging
      ↓
Molecular sampling
      ↓
Computational analysis
      ↓
Patient functional profile

3. What is the real cost of the complete system?

Replacing Matrigel and some recombinant growth factors could reduce costs, but other components of organoid culture, such as specialized media and supplements including B-27, may remain significant expenses.

Therefore, the actual cost per patient and per experimental condition is still unknown and would need to be determined experimentally after the system is optimized.

4. How reproducible is the system between patients?

Patient-derived organoids are expected to show biological variability. The platform therefore needs to distinguish between:

• Technical variability caused by the culture system
• Experimental variability caused by handling or automation
• Biological variability between patients
• Disease-associated phenotypes

The objective is not to eliminate patient variability, but to make the experimental system sufficiently standardized that biological differences can be measured reliably.

5. Can the complete workflow be scaled?

The final unknown is whether the combination of synthetic ECM, patient-derived organoids, automated culture, imaging, and molecular analysis can maintain reproducibility when the number of patients and experimental conditions increases.

The initial 50-patient/year target would therefore function as a proof-of-concept scale. If successful, the same platform could subsequently be expanded to larger patient cohorts and higher-throughput experiments, including systematic drug screening and treatment-response profiling.