Fluctuations in medium nutrient levels contributed to limitations in cell expansion.

<p>Glucose measurements from β-TC6 cell spheroids cultured in (A) Static cultures, and (B) stirred suspension bioreactors using high (4.5 g/L), intermediate (2.75 g/L), and low (1.0 g/L) glucose medium, as depicted by their position on the Y axis. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (C) No difference was seen comparing static to SSB cultures with any of the glucose levels. Comparison of expansion of β-TC6 spheroid cultures indicated that changing the glucose in the medium to achieve levels closer to the physiological range did not significantly improve cell expansion. (*indicates a p value of 0.027 compared with the same culture method using high glucose medium.) SSB: stirred suspension bioreactor.</p

Stirred suspension bioreactors offered no growth improvement compared to static cultures.

<p>Fold expansion of β-TC6 spheroids after twenty one days compared stirred suspension bioreactor to static culture using standard high glucose medium.</p

Adjusting culture medium feed rate regulates glucose concentrations and improves cell growth compared to continuous feeding at a constant rate.

<p>(<b>A</b>) Cell counts comparing constant feed rate to adjusted feed rate from the same cultures (*represents a p value <0.05 indicating a significant difference in culture expansion). (<b>B</b>) Average culture medium glucose levels from 21 day constant feeding, and adjusted feeding bioreactor cultures. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements).</p

Continuously fed SSB eliminated glucose fluctuations and improved cell expansion.

<p>(<b>A</b>) Glucose measurements for β-TC6 spheroid culture medium using static, SSB, and CF-SSB culture methods and feeding with standard high glucose medium. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (<b>B</b>) Fold Expansion of β-TC6 spheroids over 21 days of culture comparing static, SSB, and CF culture methods. SSB: stirred suspension bioreactor. CF: Continuously fed stirred suspension bioreactor.</p

Design of continuous feed apparatus.

<p>(<b>A</b>) Schematic diagram of continuous feeding perfusion circuit with fresh medium, perfusion pump, outflow tube, and waste medium. Solid blue lines are medium connections, dashed green lines are fiber optic connections, and dotted black lines are electronic connections. (<b>B</b>) Exploded illustration of outflow tube showing small pores, which do not allow spheroids to pass through, but allow the removal of waste medium. The continuous feeding system was designed to help improve consistency of nutrient and supplement concentrations making β-TC6 cell culture parameters more stable and controlled.</p

Continuous feeding at a constant rate cannot maintain a physiological glucose level.

<p>Culture medium glucose measurements from continuous fed cultures with high, intermediate, or low glucose medium during 21 days of expansion. The physiological glucose range is indicated by the grey bar.</p