3D Cultured Spheroid Models

Paradigm Shifts from Animal Testing to Alternative Preclinical Drug Screening

• Animal testing

  

  Costly, time consuming, low throughputAnimal mechanistic informationExtrapolation of results to human: 50% accuracy

  Alternative testing for preclinical drug screening

  

  The 3R principle (Reduction, Replacement, Refinement)New Approach Methodologies (NAMs)Human-based in vitro high throughput screening (HTS) assaysIn vitro cell models; In chemico studies; In silico approaches; Multi-omics analyses

3D Cultures: A Better Tool for In Vitro Cell-Based Assays

• What is 3D Cell Culture:
  3D cell culture is a commonly used method for culturing cell spheroids, representing matrix-free 3D cell culture.
  

  Type of culture	2D	3D
  In vivo-like	No mimicry of natural tissue or tumor structure	Resembles in vivo tissues and organs
  Proliferation	Faster growth in monolayer	Similar to in vivo situation
  Polarity	Partial polarization	Accurate cell polarization depiction
  Cell morphology	Sheet-like, flat cells	Form aggregate/spheroid structures
  Cell-cell interaction	Limited interactions	Proper cell-cell and cell-extracellular matrix interactions
  Gene/protein expression	Altered gene expression	Relevant gene/protein expression in 3D models
  Drug responses	Lack of correlation with human tumors	Similar drug resistance patterns as in patients
  Access to compounds	Unlimited access to nutrients and molecules	Variable access similar to in vivo conditions
  Testing duration	Short-term acute toxicity assessment	Long-term low-dose repetitive exposure

Utilization of 3D Cultured Scaffold-Free Spheroids in Preclinical Studies

• The Applications of 3D Cultured Scaffold-Free Spheroids in Preclinical Studies
  Drug screening and efficacy testingBetter predictive value for drug response compared to 2D culture systems.Evaluate toxicity, drug penetration, and therapeutic effectiveness.Disease modeling and understanding:Mimic the pathological features of diseases.Study disease progression and mechanisms.Tissue engineering and regenerative medicine:Develop bioengineered tissues for transplantation and organ-on-a-chip systems.Testing biocompatibility and functionality of engineered constructs.
  The Advantages of 3D Cultured Scaffold-Free Spheroids in Preclinical Studies
  Improved physiological relevance.Reduction in animal experimentation.Cost-effective and high-throughput screening.

Medicilon's Breakthrough: 3D Cultured Spheroids for Advanced Drug Safety Evaluation

• Drug toxicity screening and efficacy testing:
  

  Harnessing the Power of 3D Cultured KRAS Spheroids for Cancer Drug Efficacy Evaluation

  

  Upgrading the In Vitro Liver Toxicity Assessment with the 3D Cultured HepG2 Cell Spheroids Platform

Harnessing the Power of 3D Cultured KRAS Spheroids for Cancer Drug Efficacy Evaluation

• 
  KRAS mutations are frequently observed in various cancers and have been linked to cancer development, poor prognosis, and increased resistance.
• Empowering Preclinical Studies: Ready-to-Use KRAS 3D Spheroid Models Developed in Medicilon

  Overview of 3D Spheroid Models Developed with KRAS Cell Lines and Associated Diseases
  Cell line	Full Name	Organ	KRAS mutant
  SW837	Human colorectal cancer cells	Colon	G12C
  HCT15	Human colorectal cancer cells	Colon	G13D
  LoVo	Human colorectal cancer cells	Colon	G13D
  HCT116	Human colorectal cancer cells	Colon	G13D
  NCI-H23	Human non-small cell lung cancer cells	Lung	G12C
  Calu-1	Human lung cancer cells	Lung	G12C
  NCI-H358	Human non-small cell lung cancer cells	Lung	G12C
  HPAF-II	Human pancreatic cancer cells	Pancreas	G12D
  AsPC-1	Human pancreatic adenocarcinoma	Pancreas	G12D

• Evaluating the Impact of KRAS Mutations on In Vitro Colon Cell Models Exposed to 5-Fluorouracil
  
  The study utilized four colon KRAS mutation cell lines to evaluate the comparative 5FU exposure results. The IC50 values of both 2D and 3D cultured cells were measured by ATP assay at various time points (24, 48, 96, and 168 hours), providing valuable insights into the efficacy of 5FU treatment in different cell culture models.
• Investigation of Drug Effects on In Vitro NSCLC Cell Models with G12C KRAS Mutations
  
  The study investigated the effects of doxorubicin (DOX), gemcitabine, and AMG510 on multiple in vitro non-small cell lung cancer (NSCLC) cell models carrying the G12C mutation within the KRAS gene.IC50 values were determined based on the ATP assay results to evaluate treatment efficacy, representing the concentration at which 50% inhibition of cell viability was achieved after DOX, gemcitabine, or AMG510 exposure at various time points.
• Investigating Drug Effects on In Vitro Pancreatic Cancer Cell Models with G12D KRAS Mutation
  
  This study analyzed the impact of doxorubicin (DOX) and gemcitabine on two in vitro pancreatic cancer cell models carrying the G12D KRAS mutation.IC50 values were derived from ATP assay-based cell viability results and compared between 2D and 3D cultured cells across different time intervals.

Unlocking the Potential of 3D Cultured HepG2 Spheroids for Liver Toxicity Assessment in Drug Development

• 
• Experimental Workflow: Assessing Acetaminophen-Induced Toxicity Using 2D and 3D Cultured HepG2 Cells
  
• Morphological Changes in 3D Cultured HepG2 Cells after Repetitive Exposures
  
  
  
  Acetaminophen exposure to 3D HepG2 spheroids resulted in dose-dependent morphological changes, with higher concentrations inducing shrinkage.Quantitative analysis confirmed concentration-dependent inhibition of spheroid growth above 1 mM.
• Time and Dose-dependent Changes in ATP Viability and IC50 Shift
  
  ATP viability assay showed dose and time-dependent effects, with 0.5 mM initially stimulating growth but declining after 96 hours.
  
  IC50 values decreased with prolonged exposure, emphasizing the importance of extended assessment for cytotoxic evaluation.
• Pattern Shifts in Size and Viability of Long-term Exposed 3D HepG2 Cultures
  
  Hierarchical clustering analysis revealed time-dependent effects on morphology and viability in 3D HepG2 cultures.Clustering identified distinct separations, particularly at lower to middle doses, and 1 mM exposure aligned with control groups.
• Positive Correlation between Imaging Data and Viability at Each Time Point
  
  Correlation analysis between imaging data and viability consistently showed a strong positive relationship at each time point.The significant and reliable correlation between imaging parameters and cellular health confirms their potential as indicators of cellular function.

Conclusion: Long-term repetitive exposure in 3D cultured HepG2 spheroids accurately mirrors the in vivo scenario

• 

  3D HepG2 cells exhibited greater sensitivity to Acetaminophen compared to 2D cells, with significantly lower IC50 values.The close alignment between 3D model's IC50 value and clinical thresholds emphasizes the importance of using physiologically relevant models in toxicity assessments.

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