Poster M18 - Engineering an Ecosystem For Recycling Spent Cell Culture Media using Chlorella Sorokiniana

Introduction:

Introduction: Due to their costs and availability, many currently marketed advanced therapies, such as cell and antibody therapies and
tissue engineered constructs are limited in their access to large populations. Additionally, the realization of whole-organ engineered
constructs or cultured meat will depend on affordable manufacturing of large quantities of cells. As cells grow, metabolic waste, such
as ammonia, accumulates in the cell culture media to the detriment of critical cellular functions, including proliferation. It is estimated
that 300 million liters of cell culture media, a costly input, are discarded per year [1]. Spent media may be up-cyclable if metabolic
waste products are sequestered and key media components are replenished. We hypothesized that Chlorella Sorokiniana, a
thermoresistant strain of unicellular algae, can grow in spent animal cell culture media at 37°C, in the absence of light, and remove
ammonia waste to improve media use efficiency.

Materials and

Methods: Spent media stock was collected from QM7 cell cultures (a quail myoblast cell line) containing fresh media
(GM) (DMEM/F12, 10% FBS, and 1% Pen/Strep) after 4 and 8 days (4D-SGM and 8D-SGM, respectively). C. sorokiniana was
inoculated into fresh GM, 4D-SGM, and 8D-SGM at 37C, with or without light, and growth curves were generated via absorbance
measurement. Ammonia and glucose consumption curves from 4D-SGM were generated via enzymatic assays. An MTT assay was
used to assess QM7 metabolic activity in algal-treated, 4D-SGM, and fresh GM.

Results, Conclusions, and Discussions:

Results: C. sorokiniana exhibited the fastest growth in fresh GM when exposed to minimal or no light, indicating its capacity for metabolic use of
glucose independent from sunlight (Fig 1A). Greater algal growth was observed in both 4D-SGM and 8D-SGM than in fresh GM after
7 days (Fig 1B). This is believed to be due to the increased ammonia concentrations in spent media. A high density of algae (10 8
algae/ml) removed nearly all ammonia from 4D-SGM within 24 hours of treatment, and due to light independent metabolism,
consumed nearly all remaining glucose within 48 hours of treatment. Greater metabolic activity of QM7 cells was observed in algal
treated 8D-SGM compared to untreated 8D-SGM.

Conclusions: We identified the rates at which C. sorokiniana can sequester ammonia waste and we compared the metabolic activity
of animal cells grown in fresh, spent, and algal-treated spent media. By incorporating algae into mass-bioproduction systems for
animal cells, aimed at reducing the cost of the most expensive input, cell culture media, we move closer to developing an engineered
ecosystem for enhancing bioproduction capacity, reducing costs, and ultimately improving equity of next generation medicines.

Acknowledgements (Optional): Acknowledgements: This research was partially funded by a New Harvest fellowship awarded to Richard Thyden
[1] Lynch CD, et al. Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently
supports production of recombinant protein by Escherichia coli. PLoS One. 2022 May 4;17(5):e0266921. doi:
10.1371/journal.pone.0266921.