3-D Cell Culture

Know these terms !! Extra Cellular Matrix ( ECM ) Extra cellular molecules secreted by cells for mechanical support, adhesion of cells, cell to cell interaction etc… Basement membrane is an ECM

Scaffolding A temporary structure made of wooden planks or bamboo made while construction of building

Scaffolding in Biology It is used for Tissue regeneration and in-vitro drug testing. Scaffolding of human ear

A 3-D, biodegradable, synthetic scaffold molded into precise shape of hip joint. Known as Ghost heart + stem cell of recipient Functional heart Bioreactor

2-D Cell Culture Allows growth only in 2 dimensions in petridish ( mono layered ) Eg: Bacterial or yeast culture we do in our labs Yeast transformation Phage titration

Then why should we develop 3-D cell culture. Advantages Easier environmental control, cell observation and measurements Cheaper to cultivate Highly efficient in studying unicellular organisms Then why should we develop 3-D cell culture. In simple words, all our ( Human ) & animal cells are in 3-D environment. Culturing these cells in 2-D monolayer manner is a poor model for in vivo conditions and behaviors. ( Why ???!! )

Can tumor cells grown in 2-D mimic these in-vivo conditions ? Different regions of a Tumor spheroid Can tumor cells grown in 2-D mimic these in-vivo conditions ?

Let’s see an example !!! Human epithelial carcinoma cells ( A431.H9 cells ) 5-fluorouracil ( 5 FU) Grown in 2-D as well as 3-D conditions Treated with 2 anti cancer drugs with diff. activity profiles Tirapazamine ( TPZ )

Which result ( 2D or 3D ) is more reliable ??? Observation :A431.H9 cells are more resistant to 5-FU in 3D conditions than 2D TPZ in 2D conditions than 3D Which result ( 2D or 3D ) is more reliable ???

Why 2-D is a poor model for studying animals ? Mono layered ECM Cell to cell interaction Absent Cell & matrix interaction Cells cannot attain their in vivo configurations as in tissues Due to these reasons 2D cells gives inaccurate data regarding predicted response of cancer cells to chemotherapeutics.  In 2011 alone, out of approximately 900 anti-cancer therapies in clinical trials or under Federal Drug Administration review, only 12 achieved approval ; resulting in the loss of hundreds of millions of dollars that were spent on pre-clinical and clinical trials. 

3-D Cell culture models Scaffold based Non Scaffold based Polymeric Hard Scaffolds Biological scaffolds Micropatterened surface micro plates Hanging drop micro plates Spheroid micro plates with Ultra low attachment (ULA ) coating Microfluidic 3D cell culture

Commonly we use Poly styrene , Polycaprolactone Polymeric Hard Scaffolds 3-D culture made by growing cells on pre fabricated scaffolds made of polymers that mimic ECM. Commonly we use Poly styrene , Polycaprolactone

Scaffolds created of components of biological origin. Biological Scaffolds Scaffolds created of components of biological origin. Eg: fibronectin, collagen, laminin, gelatin Collagen matrix Silk Scaffold Micro patterned surface micro plates (A) round, (B) square, or (C) slit patterning within the plate well

What determines the size of spheroids ?? Hanging drop micro plates Cells in absence of a substrate to attach with will self assemble to form spheroid structure HDP top and bottom openings What determines the size of spheroids ??

Spheroid micro plates with ULA coating These plates can contain larger volume of medium than HDP Microfluidic 3D cell culture Accounts complexities such as continuous nutrient flow, waste removal etc…

Can you imagine some other method ???

Questions to Ponder… What are some challenges / limitations of 3D cell culture? Can traditional assays developed for 2D models be applied in 3D? Does humans/ animals have higher dimension than 3D ?

Bibliography www.biotek.com https://www.ncbi.nlm.nih.gov/ www.springer.com

Vishnu Narayanan S S BS-MS 2015