Chapter Four · failure evidence

What Cell Culture & Co-Culture Engineering got wrong, from 70 dissertations

The records document engineering and methodological failures encountered during the development of monoculture, co-culture, and three-dimensional tissue platforms. Common bottlenecks include synthetic consortia underperforming single strains, incompatible culture media and microenvironmental substrates, and simplified in vitro models failing to mimic physiological behavior. These records come from PhD theses at 25 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.

Three-dimensional hydrogels and organoid cultures underperformed simpler two-dimensional systems and suffered from technical bottlenecks

15 theses · 10 institutions

Researchers found that three-dimensional scaffolds and organoid systems frequently exhibited lower metabolic output, reduced cell viability, and uncontrolled growth compared to two-dimensional baselines. These systems were often abandoned or replaced due to low establishment rates, labor-intensive maintenance, and difficult cell or nucleic acid retrieval.

Tried and failed

static 3D hydrogel cell culture applied to modeling in vivo cellular secretomes. Outcome: did not generalise. Reason: Without dynamic fluid perfusion, static 3D cultures phenotypically clustered with 2D static cultures rather than in vivo models

High-throughput 3D on-chip potency assay for cell therapy products · Georgia Tech

Tried and failed

partial rather than complete media exchange applied to 3D primary leukaemia cell culture. Outcome: no signal. Reason: did not improve blast retention or fold expansion over long-term culture

Towards personalised medicine - developing a 3D leukaemia biomimicry · Imperial

Lost to a baseline

3D differentiation in all STH formulations resulted in significantly lower O4+ oligodendrocytes (STH-2: 13.63%, STH-3: 17.30%) compared to 2D laminin culture baseline (23.24%).

Towards Improved Myelination: Engineering Bioinstructive Scaffolds for Schwann Cell Differentiation and Shear-Thinning Hydrogels for Oligodendrocyte Progenitor Therapy · DSpace at SUNY Buffalo

Lost to a baseline

2D cultures displayed significantly higher resazurin turnover and higher initial AP activity (~1.6 pmol/min vs 0.2–0.4 pmol/min) compared to 3D cryogel cultures

Osteogene Differenzierung von mesenchymalen Stammzellen des Fettgewebes auf vier verschiedenen 3D-Trägermaterialien im Vergleich zu einer 2D-Kultur · Publikationssystem UB Tuebingen

Lost to a baseline

2D IFN-γ and 2D control cultures maintained higher cell viability (98.0% and 98.7%) compared to microfluidic IFN-γ culture (70.9%).

High-throughput 3D on-chip potency assay for cell therapy products · Georgia Tech

Lost to a baseline

2D monolayer culture produced substantially higher urea (up to 3.120 µg/10^6 cells/h) compared to 3D bioreactor culture (max 0.502 µg/10^6 cells/h).

Wiederbesiedlung einer azellularisierten Lebermatrix mit primären porcinen Hepatozyten sowie Kultivierung in einem Bioreaktor · Publikationssystem UB Tuebingen

Lost to a baseline

2D monolayer demonstrated active galactose consumption (up to 1.72 mg/10^6 cells in first 24 h) whereas bioreactor 3D cultures showed zero elimination.

Wiederbesiedlung einer azellularisierten Lebermatrix mit primären porcinen Hepatozyten sowie Kultivierung in einem Bioreaktor · Publikationssystem UB Tuebingen

Considered and rejected

Considered and rejected: 3D ECM scaffold construct culture for primary AML assays was rejected/bypassed in favour of a 2D thin-film ECM scaffold due to low cellular recovery and retrieval issues from dense 3D pores.

Modelling Bone Marrow Extracellular Matrix Interactions Reveal CD36-Dependant Vulnerabilities in Acute Myeloid Leukemia · YorkSpace

Considered and rejected

Considered and rejected: Rejected 3D cell culture in favor of 2.5D culture due to imaging complexity, difficult nucleic acid/protein retrieval, and non-reproducibility issues.

Combining extracellular matrix hydrogels and electrical stimulation to influence macrophage polarisation · University of Nottingham Repository

Considered and rejected

Considered and rejected: Rejected 3D organoid/microsphere culture protocols in favor of 2D monolayer cultures to enable high-throughput screening and automated immunocytochemical quantification, despite lower otic cell yields.

Erzeugung von otischen Vorläuferzellen aus humanen induzierten pluripotenten Stammzellen · Publikationssystem UB Tuebingen

Considered and rejected

Considered and rejected: 3D spheroid co-culture was abandoned in favor of 2D culture because spheroid formation and macrophage integration varied dramatically across lung cancer cell lines and was too labor intensive.

Exploiting Multi-Cell Type Cultures to Elucidate Tumor Cell Features That Impact Macrophage Phenotype · DSpace at UTSWMED

Considered and rejected

Considered and rejected: 3D brain assembloids deferred in favor of 2D mixed neural cultures for scalability and cost-effectiveness in proof-of-concept testing.

ApoE isoforms modulate SARS-CoV-2 neurological infection and confer differential risks for infection-driven early AD pathogenesis through vascular injury · JScholarship

Tried and failed

layered hydrogel sandwich culture applied to in vitro organoid growth regulation. Outcome: unstable. Reason: stem cell zones lacked intrinsic feedback control, resulting in uncontrolled exponential fission unlike in vivo

Tissue intrinsic properties of regeneration in homeostasis and injury in the mammalian small intestine · Harvard

Tried and failed

organoid co-culture for disease modeling applied to patient-derived glioma transcriptional states. Outcome: worse than baseline. Reason: suspected genetic drift prevented recapitulation of patient canonical transcriptional meta-programs

Human Brain Organoids for Studying Malignant Cell States and Intercellular Communication in Human Glioma · MIT

Lost to a baseline

Established organoid culture success rate reached only ~10% (>= passage 5), underperforming the 50% reported by Mullenders et al. (2019) and 70% (>= passage 6) by Lee et al. (2018)

Veränderungen von Expressionsmerkmalen in Blasentumor-Organoiden während Langzeitkultivierungen · Publikationssystem UB Tuebingen

Tried and failed

decellularised tissue extracellular matrix hydrogels applied to organoid cell culture. Outcome: worse than baseline. Reason: demonstrated inferior performance for organoid culture compared to standard reconstituted basement membrane matrix

Extracellular matrix hydrogels for endometrial regeneration · Imperial

Considered and rejected

Considered and rejected: Abandoned human intestinal organoid culture after repeated protocol and media modifications failed across four patient tissue preparations, switching to established murine organoids.

Der Einfluss von Teduglutid auf die epitheliale Homöostase im Dünn- und Dickdarm · Publikationssystem UB Tuebingen

Engineered microbial consortia and division of labor co-cultures underperformed single-strain monocultures

6 theses · 5 institutions

Multispecies co-cultures and divided synthetic consortia showed lower biomass accumulation, reduced catabolic enzyme activity, and diminished substrate degradation compared to single-strain baselines. Increasing heterologous gene copy numbers or co-culturing diverse strains failed to surpass the growth and metabolic performance of isolated monocultures.

Tried and failed

bacterial coculture for biodegradation applied to single-compound xylene degradation. Outcome: worse than baseline. Reason: coculture exhibited lower removal efficiency than single-strain monoculture

Study of a Bacterial Coculture for Benzene, Toluene, Ethylbenzene and Xylene Degradation · YorkSpace

Tried and failed

division of labour in microbial consortia applied to stress tolerance in engineered yeast. Outcome: worse than baseline. Reason: consortia exhibited lower growth and reduced stress tolerance compared to co-expressing monocultures under oxidative stress

Engineering division of labour in Yarrowia lipolytica to relieve the metabolic burden of polysaccharide degradation · Imperial

Tried and failed

synthetic microbial consortium division of labour applied to heterologous polysaccharide degradation in yeast. Outcome: worse than baseline. Reason: two-strain consortium showed significantly lower growth than single-strain co-expression monocultures

Engineering division of labour in Yarrowia lipolytica to relieve the metabolic burden of polysaccharide degradation · Imperial

Tried and failed

bacterial co-culture fermentation applied to enhancing antifungal metabolite production. Outcome: no signal. Reason: co-culturing multiple strains did not increase antifungal activity compared to the top single strain

Metabolites of lactic acid bacteria (Labs) from Malaysian fermented foods and a streptomyces SP. for control of fungal growth and mycotoxin production. · Cranfield

Tried and failed

increasing heterologous gene copy number applied to division of labour microbial consortia. Outcome: worse than baseline. Reason: increasing copy numbers did not increase the growth advantage of the consortium over the co-expressing monoculture

Engineering division of labour in Yarrowia lipolytica to relieve the metabolic burden of polysaccharide degradation · Imperial

Tried and failed

bacterial coculture for biodegradation enzyme induction applied to aromatic hydrocarbon degradation. Outcome: worse than baseline. Reason: coculture exhibited several-fold lower catabolic enzyme activity compared to single-strain baselines

Study of a Bacterial Coculture for Benzene, Toluene, Ethylbenzene and Xylene Degradation · YorkSpace

Lost to a baseline

Bioconversion in bacterial co-cultures (1.32 ppm) underperformed single isolated cultures of E. cloacae IFVB (4.35 ppm).

Estudio del comportamiento de bacterias aisladas de vainas de V. planifolia Jacks. ex Andrews en procesos asociados a la producción de compuestos aromáticos · Repositorio Institucional BUAP

Lost to a baseline

F1 & WT co-culture had a lower AUC (5162.25) than the halved F1 mono-culture benchmark (5807.11) at 10 mM succinate.

Investigating the role of chemotaxis in the natural source zone depletion of petroleum hydrocarbons · Imperial

Lost to a baseline

Single ethylbenzene degradation achieved 85% removal by coculture, slightly beaten by S. fonticola (97%) and M. esteraromaticum (92%) monocultures.

Study of a Bacterial Coculture for Benzene, Toluene, Ethylbenzene and Xylene Degradation · YorkSpace

Lost to a baseline

Single xylene degradation by coculture removed 14 mg/L (62%), losing to M. esteraromaticum monoculture alone which achieved 22.5 mg/L removal (87-88%).

Study of a Bacterial Coculture for Benzene, Toluene, Ethylbenzene and Xylene Degradation · YorkSpace

Lost to a baseline

Autotrophic and mixotrophic microalgal consortia reached a final biomass density almost two-thirds lower than the Chlorella vulgaris monoculture (density <0.4 g/L vs >0.4 g/L, p<0.05).

Contaminants of Emerging Concern Treatment by Microalgae: The Status and Future of Phycoremediation · Queens University Institutional Repository

Lost to a baseline

Single-amylase strain Y8TO1 outperformed both the double-amylase monoculture Y8TT18TO1 and the consortium Y8TT1+Y8TO1 in OD600 at 18 and 24 hours in flasks.

Engineering division of labour in Yarrowia lipolytica to relieve the metabolic burden of polysaccharide degradation · Imperial

Lost to a baseline

F1 & cheA co-culture had lower area under curve (AUC 8594.99) than the F1 mono-culture baseline (AUC 11614.22) at 10 mM succinate due to cell collisions or competition.

Investigating the role of chemotaxis in the natural source zone depletion of petroleum hydrocarbons · Imperial

Lost to a baseline

In 2% starch (no xylan), the monoculture Y8TO1TA1XC achieved higher OD600 (24.49) than all three DOL consortia (generalist 14.27, orthogonal 15.10, cross-feeding 16.33).

Engineering division of labour in Yarrowia lipolytica to relieve the metabolic burden of polysaccharide degradation · Imperial

Lost to a baseline

Double-amylase monoculture YTG18TO1 outperformed the YTG1+Y8TO1 consortium in growth rate (0.226 hr-1 vs 0.095 hr-1) and carrying capacity (45.467 vs 21.434 OD600).

Engineering division of labour in Yarrowia lipolytica to relieve the metabolic burden of polysaccharide degradation · Imperial

Lost to a baseline

Single-amylase strain YTO1 outperformed both the double-amylase monoculture YTT3TO1 and the consortium YTT3+YTO1 in OD600 at 24 hours.

Engineering division of labour in Yarrowia lipolytica to relieve the metabolic burden of polysaccharide degradation · Imperial

Competitive cell overgrowth and substrate coating incompatibilities disrupted co-culture organization

15 theses · 9 institutions

Culturing multiple cell types together frequently led to one population physically outcompeting or suffocating partner cells through spatial overcrowding and unconstrained proliferation. Co-culture approaches also failed when partner cells required mutually incompatible extracellular matrix coatings or disrupted monolayer attachment during sequential seeding.

Tried and failed

multi-node spatial aggregation of mixed neural cultures applied to engineered in vitro neural circuits. Outcome: worse than baseline. Reason: astrocytes exhibited interdigitation instead of domain separation, attenuating long-distance axonal fasciculation across nodes

Tissue Engineered Multi-Aggregate Cortical-Hippocampal Neural Networks for Pharmacological Investigations · Penn

Tried and failed

prolonged co-culture in matrix-producing host medium applied to microbial co-encapsulation viability. Reason: extended culture or high matrix-former inoculum killed encapsulated partner cells

Functional Materials Grown from Engineered Bacteria · Harvard

Lost to a baseline

Exiguobacter sp. (A1) preferentially grew in LSMMG in monoculture but decreased in relative population proportion over time when co-cultured in LSMMG against A2 or C

Preferential Growth of an Aquatic Bacterium in Low-Shear Modeled Microgravity · TXST Digital Repository

Considered and rejected

Considered and rejected: Rejected harvesting CN monocultures beyond D50 without astrocyte coculture due to loss of cell attachment and morphology.

Investigating selective neuronal vulnerability in Alzheimer’s and Parkinson’s disease with induced pluripotent stem cells · Oxford

Considered and rejected

Considered and rejected: Single-gel co-culture (mixing organisms in one gel) was rejected in favor of spatially separated printing due to competitive growth between species

Enhancing biomolecule production through microbial engineering and advanced manufacturing · UT Austin

Considered and rejected

Considered and rejected: Direct co-culture of macrophages with DRG neurons inside microfluidics was rejected due to incompatible substrate coating requirements (PLL only vs. PLL+laminin).

Modelling changes in excitability of the peripheral nervous system using compartmentalised microfluidic culture · University of Nottingham Repository

Considered and rejected

Considered and rejected: Rejected initial triple-culture strategy of plating DSEB astrocytes first with mitotic inhibitor AraC due to astrocyte overgrowth and poor neuronal adhesion.

Define the RelationSHIP: INPP5D/SHIP1 regulates inflammasome activation in human microglia · Harvard

Considered and rejected

Considered and rejected: Rejected 2D direct contact co-culture on Transwell membranes or glass because pericytes outcompete BMECs for surface attachment and physically disrupt monolayer confluence

MODELING THE HEALTHY AND DISEASED BLOOD-BRAIN BARRIER WITH IPSC-DERIVED VASCULAR CELLS · JScholarship

Considered and rejected

Considered and rejected: Excluded co-culturing primary glia during NGN2 neuron differentiation to avoid culture variability and batch effects introduced by primary glia.

CRISPR-based functional genomics to study gene regulatory architecture and consequences of genetic variation · ResearchWorks

Tried and failed

high-density in vitro cell culture seeding applied to T-cell enrichment and expansion. Outcome: worse than baseline. Reason: overcrowding and nutrient competition restricted efficient enrichment and growth

Materials to enable and enhance T-cell immunotherapies · Harvard

Tried and failed

high-density cell encapsulation in hydrogel scaffolds applied to 3D tissue culture scaffold. Reason: Spatial constraints and cell crowding prevented cellular proliferation and growth within the matrix.

Development of a three-dimensional in vitro vascularized human skin equivalent employing collagen/keratin hydrogels · UT Austin

Tried and failed

seeding aggregates onto preformed stromal cell beds applied to organoid co-culture in hydrogels. Outcome: worse than baseline. Reason: preformed cell bed prevented matrix integration and hindered differentiation

Vascularized, Immune-Infiltrated, and Perfusable Kidney Organoid-on-Chip Models · Harvard

Tried and failed

sequential dual-layer cell seeding with intervening coating applied to transwell co-culture barrier models. Reason: subsequent extracellular matrix coating and temporary media deprivation caused death of the pre-seeded cell layer

Mesenchymal Stromal Cell Manufacturing for the Identification of Immunomodulatory Properties In Acute Respiratory Distress Syndrome (ARDS) · Georgia Tech

Considered and rejected

Considered and rejected: Direct in situ (IS) photo-polymerization of MSC hydrogels inside cartilage defects without pre-culture, rejected because of severely restricted nutrient diffusion and host cell competition causing poor maturation

Optimization and Translation of MSC-Based Hyaluronic Acid Hydrogels for Cartilage Repair · Penn

Considered and rejected

Considered and rejected: Original 3D indirect co-culture (MSCs in insert, spheroids at bottom) was rejected because hMSCs aggregated on PET membranes and spheroids merged into a single necrotic mass.

Human rather than mouse mesenchymal stem cells in 3D co-culture models boost proliferative and migratory characteristics of colorectal cancer cells through paracrine signalling pathways, highlighting species disconnects · University of Nottingham Repository

Incompatible culture media formulations and chemical environments compromised cell viability and growth

13 theses · 10 institutions

Cells suffered reduced viability, morphology loss, and detachment when cultured in media formulations that failed to satisfy the divergent nutrient or serum demands of distinct cell types. Growth was also suppressed by toxic leachates from fabrication materials, metabolic waste accumulation in static systems, and antimicrobial treatments.

Tried and failed

standard culture medium with generic supplements applied to multi-cell neural transwell co-cultures. Outcome: worse than baseline. Reason: unoptimized astrocyte supplements compromised cell morphology and viability

Regulation of intercellular communication by Alzheimer's disease genetic risk factors · Harvard

Tried and failed

minimal medium with organic solvent carrier applied to microbial co-culture growth. Outcome: worse than baseline. Reason: nutrient starvation caused substantially slower microbial growth compared to expected baseline literature values

Determining an effective method to select and observe an algae-bacteria symbiosis to bioremediate fluoranthene · UT Austin

Considered and rejected

Considered and rejected: Pure neuronal cultures for Sholl dendritic analysis were rejected because neurons required serum-free media whereas co-cultured B cells required serum-rich media; reverted to mixed cortical cultures.

BDNF-Producing B Cells Mediate Plasticity in the Recovering Brain After Stroke · DSpace at UTSWMED

Considered and rejected

Considered and rejected: Rejected culture media with 0%, 1%, and 2.5% FBS for FLS co-culture due to observed cell death, poor adhesion, and cell detachment; adopted supplemented Neurobasal medium with 5% FBS instead.

Biomechanical & Biochemical Contributions Of Matrix Metalloproteinases In Joint Pain: Models, Mechanisms, & Patients · Penn

Considered and rejected

Considered and rejected: Culturing meningeal lymphatic co-cultures in AIM-V (supplemented with VEGF-C and EGF) or Neurobasal media, rejected due to significantly decreased LEC viability compared to Vasculife.

Engineered models of the lymphatic stroma to study cell and fluid transport · Virginia Tech

Tried and failed

photopolymer 3D printing for microfluidic cell culture applied to microbial growth chambers. Reason: resin devices released toxic leachates that inhibited cell growth

Modeling Algal Growth Under Controlled Microenvironment Using a Microfluidic Platform · Cornell

Tried and failed

escalating antibiotic and antimycotic treatment concentrations applied to contaminated 3D primary organoid cultures. Reason: failed to eliminate heavy infection and caused cytotoxicity leading to growth cessation or culture loss

Establishing 3D models for the study of intra-tumoural heterogeneity and response to chemotherapy in high grade serous ovarian cancer · Imperial

Considered and rejected

Considered and rejected: Rejected 100% breast cancer media concentration for co-culture studies because 50%/50% breast cancer/osteogenic media provided superior osteoblast viability while maintaining metastatic microenvironment factors.

Investigating Human Cell Influence on the Micromechanical Environment of a 3D-Printed Bone Tissue Model · Virginia Tech

Tried and failed

direct media recycling without supplementation or purification applied to mammalian cell culture. Outcome: worse than baseline. Reason: nutrient depletion and metabolic waste accumulation decreased viable cell density

Recycling and Regeneration of Spent Perfusion Media via Ion Concentration Polarization · MIT

Tried and failed

human plasma-like medium cell culture applied to colorectal cancer cell lines. Reason: failed to sustain cell line growth despite supporting bacterial co-culture

Pharmacomicrobiomics in gastrointestinal cancer: the gut microbiome as modulator of anticancer therapy efficacy and predictor of therapy outcomes · Imperial

Considered and rejected

Considered and rejected: Rejected batch style bioreactors due to accumulation of metabolic waste and inability to maintain cultures for weeks to months.

FODMAP availability modulates the influence of the microbiota on visceral afferent neuron activation · Queens University Institutional Repository

Considered and rejected

Considered and rejected: Rejected broth cultures (LB and KB broth) containing bacterial cells/supernatants for spore germination assays because media itself inhibited fungal spore germination

MANAGEMENT OF FUSARIUM BASAL ROT DISEASE OF ONION (ALLIUM CEPA L.) BY USING PLANT GROWTH PROMOTING RHIZOBACTERIA IN SEAWEED FORMULATION · DalSpace

Considered and rejected

Considered and rejected: Alternative culture media were omitted and abandoned because they failed to yield viable algal cultures compared to Bozniak's Community Medium, Modified Bristol's Solution, and habitat-matched enrichment formulations.

Limnology of select fresh water systems near the Antarctic Peninsula -- Field and laboratory studies · Virginia Tech

Two-dimensional monocultures failed to replicate complex tissue differentiation and heterotypic cellular interactions

8 theses · 5 institutions

Monolayer monocultures caused cell lines to undergo phenotypic convergence, drift, or loss of organoid-forming potential over extended culture. Isolated single-cell systems also failed to induce functional drug responses and structural sarcomeric organization that require direct cell-cell contacts and three-dimensional architecture.

Tried and failed

single cell type monoculture in vitro applied to spontaneous network bursting on microelectrode arrays. Outcome: no signal. Reason: isolated motor neurons lacked synaptic partners necessary to generate correlated spontaneous firing and bursting

Enrichment for V1 interneurons from mouse embryonic stem cells and evaluation of de novo network formation · UT Austin

Tried and failed

single-cell-type monoculture assays applied to drug-induced fluid drainage response. Outcome: no signal. Reason: functional response to drug required heterotypic cell-cell interactions present only in co-culture

Tissue-engineered in vitro models for studying lymphatic morphogenesis, pathogenesis, and drug delivery · Cornell

Tried and failed

2D monolayer cell culture applied to developmental cell phenotype differentiation. Outcome: no signal. Reason: Monolayer culture caused expression profiles to converge, masking differential gene expression present in 3D culture

Characterising embryonic stem cell-derived tenocytes and determining the changing role of scleraxis during tendon development · Cambridge

Tried and failed

organoid derivation after extended 2D monolayer culture applied to primary cancer cells. Outcome: no signal. Reason: cells maintained in 2D culture for over 24-48 hours lost the capacity to form 3D organoids

Establishing 3D models for the study of intra-tumoural heterogeneity and response to chemotherapy in high grade serous ovarian cancer · Imperial

Tried and failed

2D monolayer cell culture applied to acidic metabolite cytotoxicity screening. Reason: cells lost 50% viability under physiological lactic acid concentrations unlike 3D tissue models

Mechanistic insights into the role of bacterial metabolites on microbial-induced inflammation in vaginal epithelial cells · Imperial

Tried and failed

2D in vitro cell culture and passaging applied to primary mouse endometrial epithelial cells. Reason: poor cell viability and lack of attachment during passaging

Extracellular matrix hydrogels for endometrial regeneration · Imperial

Considered and rejected

Considered and rejected: Rejected relying on 2D human myoblast cultures alone for contractile studies, choosing 3D bioengineered microtissues/bundles to enable aligned sarcomeric organization.

Metabolic Reprogramming and Stem Cell Rejuvenation for Skeletal Muscle Regeneration after Aging · DSpace at SUNY Buffalo

Considered and rejected

Considered and rejected: Rejected using 2D monolayer culture to study developmental divergence between adult and fetal tenocytes due to phenotypic convergence/drift.

Characterising embryonic stem cell-derived tenocytes and determining the changing role of scleraxis during tendon development · Cambridge

Considered and rejected

Considered and rejected: Rejected conditioned media (indirect co-culture) approaches because proliferation enhancement required direct juxtacrine EC-MuSC contact.

INVESTIGATING VASCULAR ENDOTHELIAL CELL-MUSCLE STEM CELL COMMUNICATION IN SKELETAL MUSCLE IN VIVO AND IN VITRO · Cornell

Simplified in vitro culture environments failed to capture physiological host dynamics and in vivo behaviors

9 theses · 8 institutions

In vitro culture systems failed to reproduce host ecological conditions, resulting in mismatched microbial coexistence, loss of metabolic activity, and altered volatile profiles compared to living hosts. In vitro assays also failed to predict complex organismal processes such as systemic nanoparticle distribution and multi-lineage immune responses.

Tried and failed

monoculture susceptibility assays in nutrient-rich media applied to predicting multispecies community composition under stress. Outcome: did not generalise. Reason: In vitro single-species susceptibility tests fail to capture interspecies interactions and complex ecological dynamics in polymicrobial communities

IDENTIFYING MICROBIAL BIOMARKERS OF CYSTIC FIBROSIS HEALTH AND DISEASE · Georgia Tech

Tried and failed

in vitro high-throughput droplet co-culture assays applied to predicting in vivo microbial coexistence outcomes. Outcome: did not generalise. Reason: In vitro media failed to recapitulate host environment dynamics allowing coexistence in vivo but not in vitro

Bacterial Interspecies Interactions and Microbial Community Assembly · MIT

Tried and failed

in vitro culturing to replicate volatile emission differences applied to host-associated microbial isolates. Outcome: no signal. Reason: isolated culture plates did not reproduce differential volatile profiles observed in vivo

WHEN FLOWERS PLAY DEAD: MICROBES AS ARCHITECTS OF A ‘DECEPTIVE’ FLORAL PHENOTYPE · Cornell

Tried and failed

serial subculturing of anaerobic enrichment cultures applied to chemolithoautotrophic microbial communities. Outcome: no signal. Reason: metabolic activity failed to sustain after transfer into fresh synthetic growth media

Coupled iron reduction-ammonium oxidation (Feammox) in alkaline soils polluted with nitrogen · HARVEST

Lost to a baseline

Conventional static cell cultures fail to replicate dynamic mechanical and biochemical stimuli present in vivo compared to perfusion/rotating bioreactors

The promise of tissue engineering and regenerative medicine in the future of transplant surgery · OpenBU

Considered and rejected

Considered and rejected: Rejected relying on oral inoculation of SPF mice for in vivo Fusobacterium-fibroblast studies due to failure of stable colonization after 24 hours; switched to in vitro coculture.

Fusobacterium-Stromal Cell Interactions in Colorectal Cancer · Harvard

Considered and rejected

Considered and rejected: Rejected relying on in vitro / cell-culture nanoparticle delivery assays because they poorly predict in vivo delivery in adult animals.

Translational Design of Lipid Nanoparticles (LNPs) to Deliver mRNA Therapies to Solid Tumors and to the Lungs · Georgia Tech

Considered and rejected

Considered and rejected: Rejected using cultured monocytic cell lines (THP-1, U937) for mechanistic studies because they failed to replicate in vivo monocyte signaling and VEGF splicing.

Control of the collateral formation of cardiovascular disease by SRPK1 · University of Nottingham Repository

Considered and rejected

Considered and rejected: Rejected in vitro immune co-culture killing screen because it would fail to recapitulate full multi-lineage immune surveillance architecture.

Epigenetic contributions to cancer immunosurveillance · Imperial

Left open by the authors

Problems the authors named and did not get to.

Left open

Co-culture ROSMAP-derived astrocytes with familial Alzheimer's disease neurons to evaluate non-cell-autonomous astrocyte responses to diseased neuronal environments. Blocker: Requires wet-lab iPSC cell culture and co-culture experimental facilities.

Contribution of SORL1 in human iPSC-derived neurons and astrocytes to Alzheimer’s disease pathogenesis · Harvard

Left open

Co-culture AKO astrocytes with AKO endothelial cells or neurons in Transwell systems to measure manganese transport, BBB permeability, and neuronal survival. Blocker: Requires wet lab facilities, specific knockout mouse cell lines (AKO astrocytes/endothelial cells), and cell culture apparatus.

THE INS AND OUTS OF MANGANESE: ROLES OF ZIP14 IN BLOOD-BRAIN BARRIER TRANSPORT AND ASTROCYTE FUNCTION · Cornell

Left open

Deconvolute cell-autonomous versus non-cell-autonomous proteostatic stress effects using pure sorted MNs, interneuron/astrocyte co-cultures, and microfluidic motor unit circuits. Blocker: Requires wet lab facilities, cell culture protocols, microfluidic devices, and biological reagents

Modulating Proteostatic Stress in Human Motor Neurons to Identify Neuroprotective Targets · Harvard

Left open

Test whether C1q depletion or CD55 inhibition reverses synaptic phenotypes in CLU-deficient triple co-cultures. Blocker: Requires a wet lab with iPSC-derived neuronal, astrocytic, and microglial co-culture platforms and complement inhibition assays.

Regulation of intercellular communication by Alzheimer's disease genetic risk factors · Harvard

Left open

Investigate signaling dynamics and the effect of INPP5D-deficient microglia on human neurons using co-culture experiments. Blocker: Requires wet-lab facilities for human iPSC differentiation, neuron co-culture, and biochemical signaling assays

Define the RelationSHIP: INPP5D/SHIP1 regulates inflammasome activation in human microglia · Harvard

Left open

Elucidate the fitness contributions of the cobalamin-dependent gene cluster and Rli47 in Listeria monocytogenes during co-culture with other bacteria. Blocker: Requires wet lab microbiological experiments and co-culture assays with bacterial strains

Exploration of the transcriptomes and functional contributions of Listeria monocytogenes plasmids during food production-associated stress conditions · Iowa State

Left open

Assess the functionality and lysosomal enzyme composition of axonal versus dendritic autophagic vacuoles using microfluidic isolation chambers. Blocker: Requires a wet lab, primary neuronal cultures, microfluidic devices, and biochemical/microscopy assays.

Compartment-Specific Regulation Of Autophagy In Neurons · Penn

Left open

Test whether CLU genotype-driven phenotypes in human multi-cell co-cultures depend on an APOE4/4 microglial background versus an APOE3/3 background. Blocker: Requires a wet lab to culture and differentiate human iPSC-derived microglia and co-culture platforms.

Regulation of intercellular communication by Alzheimer's disease genetic risk factors · Harvard

Left open

Conduct pure and co-culture experiments to validate predicted direct interspecies electron transfer mechanisms. Blocker: Requires a wet lab, microbial strains, and specialized culturing apparatus.

Study of extracellular electron transfer in engineered methanogenic systems · Imperial

Left open

Perform single-cell RNA-seq on human iPSC-derived neuron, astrocyte, and microglia triple co-cultures across additional individuals. Blocker: Requires wet-lab iPSC differentiation, triple co-culture experimental models, and biological sequencing

Contribution of SORL1 in human iPSC-derived neurons and astrocytes to Alzheimer’s disease pathogenesis · Harvard

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