Combination of Flow with 3d Cell Culture Improves

Combination of flow with AD cell culture improves metabolic competency in multiple cell types Lewis ME,a* Gordon P,a Tolland F,b Camellias D,c Vinci B,c Oppressors S,b Casework E,a Darrel K,a Wilkinson KM a a.

Shirttails Ltd. , Sheffield I-J. B. Reincarnate Ltd. , Durham I-J.

C. University of Pisa, Pisa, Italy. *Corresponding author. Shirttails Ltd.

, 40 Lawgiver Rd, Sheffield, I-J, SO RD 1. Introduction Growing cells in AD under static conditions is the gold standard of cell culture.However, these conditions are not sufficiently representative of the complex in vivo environment, and encourage cells to behave differently than they would in the host organism. This discrepancy results in misleading data. Often, experimentation on cells cultured in standard static AD conditions is not sufficient to meet research requirements. In order to explore the complex interplay between cells and organs, and the effect of certain changes on those systems whether genomic, physiological or medical, animal models are required.

Animal models are thought to provide a better representation of human physiology and biochemistry than can be achieved with the rent gold standard of culture, and while true to a point, animal models cannot be relied upon for generating truly representative data. Indeed, there are many examples in the literature of highlighter failings of animal models to predict human toxicity[1-4]. As such, there is a requirement in research for alternatives to both animal models and standard in vitro cell culture. Ђ The Quasi-Vivo@ culture system enables cell culture under laminar flow. This is designed to mimic the type of flow found in the body[5],[6] providing mechanical stimulation equivalent to blood and humph movement, and ensuring constant delivery of nutrients/removal of metabolic by-products. The system recalculates, meaning that cells can condition the medium, and require medium change less frequently than cells in static plate wells.

Gas- permeable silicone allows the maintenance of pH and pop to incubator atmospheric levels. Ђ In vivo, cells interact in three dimensions. In vitro, AD culture restricts this capacity. AD scaffolds provide cells with a structure which replicates the function and architecture of the in vivo extracurricular matrix and encourages them to behave and interact as they would in vivo. Ђ Flow cell culture systems, combined with AD scaffolds such as constitute an in vitro environment which replicates the key features of the environment in vivo.These technologies enable study of biological systems consisting of multiple cell types, as well as study of human cells and tissues directly, reducing the need for costly, unreliable and ethically contentious animal experimentation in the short term, helping researchers refine their animal needs where there is still a requirement, and, in the long term, work towards replacing the animal model altogether.

The results presented here demonstrate the advantage of flow and AD ultra for various cell types.The increased sensitivity of rat primary hypotheses to kaleidoscopically under flow, and a novel optical chamber are also presented. alvetex@scaffold, E) Optical chamber enables observation of cells or tissue during culture under flow Cell observation forms part of the study of any cell activity, as it reveals changes in cell morphology and condition (e. G. Optimism) that might give further weight to quantitative experimental data. The optical chamber allows cells on standard 24 well plate coverall's to be observed (Fig 3) under flow by standard light microscopy.

120 Cell viability (% of static control)Figure 3. Validation of the optical chamber. a, b) The optical chamber prototype with observation windows in the top and bottom of the chamber, to enable viewing of cells through either inverted or upright microscope. C) INCHING human lung carcinoma cells stained with homosexuality and eosin and visualized in the chamber on glass coverlets at lox magnification. Scale bar is 400 GM.

D) INC-H2O cells at xx magnification. Scale bar is 200 GM. E) INCHING cell viability on glass coverall's cultured either in 24 well plate or optical chamber for 24 h. Viability was assessed using reassuring reduction agent. 20O Static Culture vessel Optical chamber The Quasi-Vivo@ system offers better predictive toxicology than standard static plate culture The presence of flow overcomes a number of insufficiencies associated with static cell culture. Flow provides cells with mechanical stimulation in the form of shear stress, supplies cells with fresh nutrients and removes toxic metabolic products[6].

To demonstrate the metabolic competency of cells cultured in the Quasi- Vivo@ system, freshly isolated rat hypotheses (RPR) were exposed to kaleidoscopically (a Hippocratic antiseptics drug) for 24 h after 24 h in flow or tactic conditions.RPR cultured under flow were found to have increased sensitivity to kaleidoscopically compared to those cultured under static conditions. 140 Static 2. Methods Alvetex@scaffolds: 106 INC-H2O human lung carcinoma cells were seeded in RPM at a density of 0. 2 x on glass coverall's (coated with 0.

1 MGM/ml rat tail collagen l) or alvetex@scaffolds in a 24 well plate, then coated with 1 MGM/ml collagen and transferred to Quasi-Vivo@ fabricators or a new well plate. Cells were incubated for ah, and viability was assessed by lumberman reassures reduction assay.Heaps hypothetically carcinoma ells were seeded onto alvetex@scaffolds at a density of 0. 3 x 106 either in a 24 well plate or a fabricator. Cells were incubated for 3 days under flow or static - medium was changed daily in the static controls, single-pass in the Quasi-Vivo system. After 3 days, viability was assessed by MET assay.

Levels of glucose, lactic acid and albumin levels were measured. Optical chamber validation: INC-H2O human lung carcinoma cells were seeded in RPM at a density of 0. 2 x 106 on glass coverall's (coated with 0. MGM/ml rat tail collagen l) in a 24 well plate, then coated with 1 MGM/ml allege and transferred to Quasi-Vivo@ fabricators or a new well plate. Cells were incubated for ah, and viability was assessed by lumberman reassures reduction assay.

Hypothetical screening: Rat primary hypocrite (RPR) or Upset cells were seeded onto plastic coverall's coated with 0. 1 MGM/ml rat tail collagen l. Seeded coverall's were coated with 1 MGM/ml collagen and transferred to either a Quasi-Volvo@ fabricator or a new 24 well plate, and incubated for ah. After ah systems were dismantled and coverall's transferred to the static well plate.Cells were incubated or ah in medium containing various concentrations of kaleidoscopically, and viability was assessed by lumberman reassures reduction assay.

Flow 3. Results Combining alvetex@scaffold AD scaffolds with flow in the Quasi-Volvo@ system improves cell viability and activity AD scaffolds such as alvetex@scaffolds are the next generation of seeding matrix, enabling cells to organize themselves into more complex structures with intracellular interactions that resemble those found in vivo - a powerful tool for research when combined with the advantages of flow.The performance of INC-H2O and Heaps cells on different seeding matrices was assessed. For both cell types, culture on alvetex@scaffolds under flow provided the highest level of cell viability (Fig la, Fig AAA). When cultured under these conditions, Heaps cells demonstrated the most in vivo-like hypocrite function as determined by high glucose consumption and albumin production, and low lactic acid production (Fig c-e).

The presence of flow not only increases the proliferation and improved the activity of these cells, but also encourages their migration into the scaffold (Fig b, Fig b). 00 2 3 15 Kaleidoscopically concentration (mm ) Figure 4. RPR viability on exposure to kaleidoscopically. Hypotheses were cultured under flow or static conditions for 24 h, followed by exposure to kaleidoscopically at various concentrations. Viability after this period was determined by reassures reduction assay, and cell viability was calculated relative to the static untreated control.

Bars shown are standard error. 4. Discussion AD static culture is not representative of the in vivo environment.Primary cells, especially hepatic cell types, exhibit a dramatic loss in phenotypes characteristics following isolation from an organ[7] and De-differentiate quickly when cultured as monolayer's in vitro[8,9]. Ђ Animal models enable the study of systemic responses in vivo, but come with ethical and practical considerations. Animals are not humans; research conducted using them will not always be accurate, and failures may have serious consequences[1-4].

Ђ By combining AD culture with flow, the in vivo environment can be simulated - cells are able to interact in multiple dimensions, are subject to the same shear stress and pressure present in the body, and can interact with cells of other types and organs due to the modularity of the system. a) b) Viability (% of static glass control) 250 200 150 50 O Glass, static Glass, flow Elevate, static Elevate, flow Culture condition Figure 1 . Optimization of seeding and culture conditions for INC-H2O lung carcinoma cells. a) INC-H2O viability under different culture conditions.

Cells were cultured under static or flow for ah on glass coverall's or alvetex@scaffolds, and viability determined by reassuring reduction assay. Viability was calculated relative to the glass static control. Bars shown are standard error. B) INC-H2O cell proliferation in the alvetex@scaffold.

Scaffolds were preserved, sectioned and stained with homosexuality and eosin to determine cell penetration. Scale bar is pm. Ђ The effect of combining these two important biological facets is clear - viability is improved, an advantage in proliferation and matrix penetration is gained, and hypocrite function is restored (Fig 1, 2).The presence of flow alone is sufficient to reveal RPR toxicity otherwise missed by static culture (Fig 4).

The Quasi-Vivo@ system, together with AD scaffolds such as alvetex@scaffold, provides the capability to study primary cells without fearing loss of function, and is a step towards the dual overarching goals of more representative in vitro research alongside reducing and eventually replacing animals in experiments. Acknowledgements Shirttails would like to thank the Technology Strategy Board for project funding. C) d) Figure 2.