Processing and secretion of insulin-related peptides in an insulinoma cell line

Grampp, Gustavo; Lodish, Harvey F.; Stephanopoulos, Gregory

doi:10.1002/(sici)1097-0290(19970205)53:3<283::aid-bit6>3.0.co;2-e

Cited by 1 publication

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“…The manufacturer's reported conversion between standard insulin activity units and equivalent grams of mature human insulin was approximately 27.6 μU/ng insulin. High‐performance liquid chromatography (HPLC) analysis of secreted and intracellular peptides from βTC‐3 cells confirmed that significant amounts of mature insulin and proinsulin are present in the samples (Grampp et al, 1996b). Accordingly, all RIA data reported include contributions from both species and are reported as units of total IRPs.…”

Section: Sample Preparation For Radioimmunoassay (Ria)mentioning

confidence: 95%

Cyclic Operation of Ceramic‐Matrix Animal Cell Bioreactors for Controlled Secretion of an Endocrine Hormone. A Comparison of Single‐Pass and Recycle Modes of Operation

Grampp

Applegate

Stephanopoulos

1996

Biotechnology Progress

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Controlled secretion processes for the production of secretory proteins in monolayer culture have been described previously (Grampp et al. Adv. Biochem. Eng./ Biotechnol. 1992, 46, 35-62), but little is known about the feasibility of scaling such processes into high-density bioreactors. Two immobilized-cell, ceramic-matrix bioreactor configurations were tested using the beta TC-3 cell model system which, in monolayer culture, can be manipulated to secrete murine insulin in a highly controlled manner. One reactor was configured with an external recirculation reservoir for oxygen transfer and was operated as a conventional immobilized bed/recycle reactor. The other reactor was configured as a single-pass perfusion system with oxygen supplied by diffusion from silicone tubing positioned proximal to the porous walls of the ceramic matrix. After inoculation with beta TC-3 cells, both systems were perfused with serum-supplemented medium to stimulate cell growth, and they ultimately attained high densities (approximately 5 x 10(8) cells/mL of pore volume). To initiate controlled secretion operations, the reactor cores were washed with a serum-free basal medium, then exposed to a serum-free discharging medium containing secretory stimulants. Following several hours of discharging, the reactors were washed again, then switched to a serum-containing medium designed to quench the regulated secretion process. For the single-pass reactor these cycling operations were simple to implement and were effective in promoting the cyclic discharge and recharge of murine insulin. Because of the ability to reduce the perfusion rate in the single-pass reactor independent of oxygen transfer, the discharged insulin was captured in a relatively small volume (2 reactor core hold-up volumes), yielding a mean product concentration 10-fold greater than in the steady-state perfusate. Cyclic operation of the recirculating reactor was more difficult due to the complexity of switching between recirculation reservoirs, and the introduction of air bubbles during such operations resulted in the loss of biomass from the reactor after one cycle. Even in the first discharging cycle, the insulin yield was much lower than in the perfusate from the single-pass reactor, despite the comparable metabolic rates. The single-pass reactor was cycled successfully through four discharging and recharging episodes and maintained its ability to discharge insulin, albeit at a slower rate after the first discharge. Overall, 50-60% of the insulin secreted during the 48 h cycles was recovered during the brief discharging episodes. When insulin secretion rates and discharging yields were normalized to metabolic activity, neither high-density reactor system performed as well as did identically treated control T-flask cultures. It is hypothesized that the productivity and responsiveness of the high-density, pore-immobilized beta TC-3 cells are lower than in monolayer culture.

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Section: Sample Preparation For Radioimmunoassay (Ria)mentioning

confidence: 95%