Annual IBC Cell Therapy Bioprocessing meeting has finished Yesterday. It was great meeting for me! I’d highly recommend it to everyone, who involved in different aspects of clinical cell manufacturing. The common theme of conference was transition of clinical cell culture to 3D settings. The field is coming to realization that only 3D cell culture will allow efficient industrial scaling up for many clinical applications. For some areas, such as manufacturing of allogeneic cells and bioengineering of thick tissue constructs or organ prototypes, 3D era is inevitable! For example, Athersys (reported by CSO John Harrington) calculated that industrial production of their stem cell product MultiStem, will require production from few trillions (for GVHD) to few hundred trillions (for stroke) per year. Even though, their current process (2D) can get biolliions of cells, it’s too labor-intensive (72 10-layer cell factories per lot require up to 13 technicians on seeding and harvest days) to make it affordable. Another interesting calculation was presented by Mark Szczypka (Pall Life Sciences) – he said: “In order to generate 250B mesenchymal stromal cells you need 1332 10-layer stack cell factories”.

In order to scale up to billions of cells we have to achieve exponential growth. Flat 2D cultures gives us linear growth, but 3D in suspension can give us exponential cell growth. That means transition to suspension culture should be made for adherent cells (traditionally cultured on flat surface), such as mesenchymal stromal cells (MSC). In the last few years a lot of work has been done to validate MSC culture on microcarriers in bioreactors. Based on recent achievements, we can confidently say that MSCs expansion in suspension on microcarriers is ready to move from process development stage to clinical production. Athersys invested 7 years in process development for scaling up manufacturing with lowering cost of goods. They were able to achieve good results with SoloHill microcarriers. Cell concentration at harvest day was ~0.5M/ml in 2% FBS media and ~ 0.24M/ml in serum-free media. John Harrington (Athersys) says that they didn’t see significant change of product cell characteristics (including potency) while moving from 2D to 3D. Celgene is also validating microcarriers for their placental MSCs. They got very good results with Cytodex beads. So, companies, which manufacturing therapeutic adherent cells (Athersys, Celgene) are moving to 3D suspension cultures. Pluristem made this move while ago, using their own bioreactor. Speakers from Athersys, Celgene and Pall noticed that many things should be considered in process development in transition to 3D. For example, some bioreactors don’t perform well with microcarriers and cells prefer certain type of microcarriers (Celgene was not able to grow their placental MSCs on SoloHill beads). Also, I noticed the absence of good reliable assays for detection of residual microcarriers in final product. If you do transition from 2D to 3D culture system in clinical cell manufacturing, pay attention to product comparability (before and after). Potency assay should be prioritized. Remember, it’s a huge process change and you may end up with completely different product after such transition. So, better to do it early on.

Let’s switch gears and look at organ bioengineering. Creating of thick tissues and organ prototypes is possible only in 3D. The major missing part here is cell number and cell density. Such constructs will require billions of cells. Jeff Morgan from Brown University reported a new way of manufacturing building blocks for bioengineered organs. He said that knowing how many total cells in our body (~37 trillions), cell number per organ and cell growth kinetics in culture (he mentioned achievable 20-fold expansion every 4 days), we can calculate time for “growing whole organ cells”. Thus, growing cell mass for whole heart or kidney will take ~35 days, liver ~ 40 days. His lab created non-adhesive molds (now commercialized by MicroTissues), which allow cells self-assembly to microtissues at high density (2.4 x 10e8/ml). They designed micro-molds of different shapes, allow to form cell spheroids, toroids (doughnut-shaped) and honeycombs. Few honeycombs can form large honeycomb with few millions of cells. Then they developed a device, which will grip and place honeycombs or toroids on each other analogously to additive manufacturing. These parts can be fused in one construct within 48 hours. These are building blocks for organs! About 1000 large honeycombs will be required to build whole heart. But it’s totally possible! Despite many challenges (vascularization is one of the biggest), we have now some manufacturing tools to build thick tissues and organ prototypes.