This research was funded by grants from the National Institutes of Health (R01 GM148741 I.R.S.), U01 EY034591, R01 EY034364, BrightFocus Foundation, Research to Prevent Blindness (J.R.C.) and R01 EY006641, R01 EY017863 and R21 EY032597 (J.B.H.).

Pumpless Multi-Channel Flow Culture to Maintain and Assess Tissues and Cells

<ol>
	<li>Lacy, P. E., Walker, M. M., Fink, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perifusion+of+isolated+rat+islets+in+vitro:+Participation+of+the+microtubular+system+in+the+biphasic+release+of+insulin.">Perifusion of isolated rat islets in vitro: Participation of the microtubular system in the biphasic release of insulin.</a> Diabetes. 21 (10), 987-998 (1972).</li><li>Doliba, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+and+ionic+coupling+factors+in+amino+acid-stimulated+insulin+release+in+pancreatic+beta-HC9+cells.">Metabolic and ionic coupling factors in amino acid-stimulated insulin release in pancreatic beta-HC9 cells.</a> American Journal of Physiology. Endocrinology and Metabolism. 292 (6), E1507-E1519 (2007).</li><li>Sweet, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+ATP/ADP+in+pancreatic+islets.">Regulation of ATP/ADP in pancreatic islets.</a> Diabetes. 53 (2), 401-409 (2004).</li><li>Chertov, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+glucose+in+photoreceptor+survival.">Roles of glucose in photoreceptor survival.</a> The Journal of Biological Chemistry. 286 (40), 34700-34711 (2011).</li><li>Kooragayala, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Oxygen+Consumption+in+Retina+Ex+Vivo+Demonstrates+Limited+Reserve+Capacity+of+Photoreceptor+Mitochondria.">Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria.</a> Investigative Ophthalmology &amp; Visual Science. 56 (13), 8428-8436 (2015).</li><li>Bisbach, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Succinate+Can+Shuttle+Reducing+Power+from+the+Hypoxic+Retina+to+the+O2-Rich+Pigment+Epithelium.">Succinate Can Shuttle Reducing Power from the Hypoxic Retina to the O2-Rich Pigment Epithelium.</a> Cell Reports. 31 (5), 107606 (2020).</li><li>Du, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+mitochondrial+pyruvate+transport+by+zaprinast+causes+massive+accumulation+of+aspartate+at+the+expense+of+glutamate+in+the+retina.">Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina.</a> The Journal of Biological Chemistry. 288 (50), 36129-36140 (2013).</li><li>Hass, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Succinate+metabolism+in+the+retinal+pigment+epithelium+uncouples+respiration+from+ATP+synthesis.">Succinate metabolism in the retinal pigment epithelium uncouples respiration from ATP synthesis.</a> Cell Reports. 39 (10), 110917 (2022).</li><li>Kamat, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluidics+system+for+resolving+concentration-dependent+effects+of+dissolved+gases+on+tissue+metabolism.">Fluidics system for resolving concentration-dependent effects of dissolved gases on tissue metabolism.</a> Elife. 10, e66716 (2021).</li><li>Stryer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry. , (1995).</li><li>Gu, X., Ma, Y., Liu, Y., Wan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+respiration+in+adherent+cells+by+Seahorse+XF96+Cell+Mito+Stress+Test.">Measurement of mitochondrial respiration in adherent cells by Seahorse XF96 Cell Mito Stress Test.</a> STAR Protocols. 2 (1), 100245 (2021).</li><li>Engel, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+dysfunction+in+Sorsby+patient-derived+retinal+pigment+epithelium.">Extracellular matrix dysfunction in Sorsby patient-derived retinal pigment epithelium.</a> Experimental Eye Research. 215, 108899 (2022).</li><li>Zhang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Mitochondrial+Respiration+Impairs+Nutrient+Consumption+and+Metabolite+Transport+in+Human+Retinal+Pigment+Epithelium.">Inhibition of Mitochondrial Respiration Impairs Nutrient Consumption and Metabolite Transport in Human Retinal Pigment Epithelium.</a> Journal of Proteome Research. 20 (1), 909-922 (2021).</li><li>Hurley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retina+Metabolism+and+Metabolism+in+the+Pigmented+Epithelium:+A+Busy+Intersection.">Retina Metabolism and Metabolism in the Pigmented Epithelium: A Busy Intersection.</a> Annual Review of Vision Science. 7, 665-692 (2021).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+activation+and+photoreceptor+survival+in+retinal+detachment.">Autophagy activation and photoreceptor survival in retinal detachment.</a> Experimental Eye Research. 205, 108492 (2021).</li><li>Okawa, H., Sampath, A. P., Laughlin, S. B., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+consumption+by+mammalian+rod+photoreceptors+in+darkness+and+in+light.">ATP consumption by mammalian rod photoreceptors in darkness and in light.</a> Current Biology. 18 (24), 1917-1921 (2008).</li><li>Lakkaraju, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+the+retinal+pigment+epithelium.">The cell biology of the retinal pigment epithelium.</a> Progress in Retinal and Eye Research. , 100846 (2020).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+strategies+of+engineering+retinal+organoids+and+organoid-on-a-chip+in+modeling+intraocular+drug+delivery:+Current+progress+and+future+perspectives.">Emerging strategies of engineering retinal organoids and organoid-on-a-chip in modeling intraocular drug delivery: Current progress and future perspectives.</a> Advanced Drug Delivery Reviews. 197, 114842 (2023).</li><li>Arjamaa, O., Nikinmaa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-dependent+diseases+in+the+retina:+role+of+hypoxia-inducible+factors.">Oxygen-dependent diseases in the retina: role of hypoxia-inducible factors.</a> Experimental Eye Research. 83 (3), 473-483 (2006).</li><li>Kamat, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Versatile+Multi-Channel+Fluidics+System+for+the+Maintenance+and+Real-Time+Metabolic+and+Functional+Assessment+of+Tissue+or+Cells.">A Versatile Multi-Channel Fluidics System for the Maintenance and Real-Time Metabolic and Functional Assessment of Tissue or Cells.</a> Cell Reports Methods. In Press. , (2023).</li><li>Neal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Low-Level+Drug+Effects+Using+Real-Time,+in+vitro+Measurement+of+Oxygen+Consumption+Rate.">Quantification of Low-Level Drug Effects Using Real-Time, in vitro Measurement of Oxygen Consumption Rate.</a> Toxicological Sciences. 148 (2), 594-602 (2015).</li><li>Jung, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+cytochrome+C+is+an+essential+regulator+of+sustained+insulin+secretion+by+pancreatic+islets.">Reduced cytochrome C is an essential regulator of sustained insulin secretion by pancreatic islets.</a> The Journal of Biological Chemistry. 286 (20), 17422-17434 (2011).</li><li>Rountree, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+insulin+secretion+by+cytochrome+C+and+calcium+signaling+in+islets+with+impaired+metabolism.">Control of insulin secretion by cytochrome C and calcium signaling in islets with impaired metabolism.</a> The Journal of Biological Chemistry. 289 (27), 19110-19119 (2014).</li><li>Calton, M. A., Beaulieu, M. O., Benchorin, G., Vollrath, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+measuring+extracellular+flux+from+intact+polarized+epithelial+monolayers.">Method for measuring extracellular flux from intact polarized epithelial monolayers.</a> Molecular Vision. 24, 425-433 (2018).</li><li>Jarrett, S. G., Rohrer, B., Perron, N. R., Beeson, C., Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+mitochondrial+damage+in+retinal+cells+and+tissues+using+quantitative+polymerase+chain+reaction+for+mitochondrial+DNA+damage+and+extracellular+flux+assay+for+mitochondrial+respiration+activity.">Assessment of mitochondrial damage in retinal cells and tissues using quantitative polymerase chain reaction for mitochondrial DNA damage and extracellular flux assay for mitochondrial respiration activity.</a> Methods in Molecular Biology. 935, 227-243 (2013).</li><li>Perron, N. R., Beeson, C., Rohrer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+alterations+in+mitochondrial+reserve+capacity;+a+means+to+predict+subsequent+photoreceptor+cell+death.">Early alterations in mitochondrial reserve capacity; a means to predict subsequent photoreceptor cell death.</a> Journal of Bioenergetics and Biomembranes. 45 (1-2), 101-109 (2013).</li><li>Cabrera, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-throughput+assays+for+evaluation+of+human+pancreatic+islet+function.">high-throughput assays for evaluation of human pancreatic islet function.</a> Cell Transplantation. 16 (10), 1039-1048 (2008).</li><li>Doliba, N. M., Qin, W., Vinogradov, S. A., Wilson, D. F., Matschinsky, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+acid+acutely+inhibits+acetylcholine-+but+not+GLP-1-stimulated+insulin+secretion+in+mouse+pancreatic+islets.">Palmitic acid acutely inhibits acetylcholine- but not GLP-1-stimulated insulin secretion in mouse pancreatic islets.</a> American Journal of Physiology. Endocrinology and Metabolism. 299 (3), E475-E485 (2010).</li></ol>

I.R.S., M.G., and K.B. have financial ties to EnTox Sciences, Inc. (Mercer Island, WA), the manufacturer/distributor of the BaroFuse perifusion system described in this study. All the other authors declare no conflicts of interest.

Due to the importance of bioenergetics in all aspects of cell function and maintenance of various components of the eye, there is a critical need for methods to study its regulation. In particular, neural retina and RPE depend on metabolism for both generation of energy as well as intra- and inter-cellular signaling14,15,16,17. Because of their high oxidative capacity, isolated tissues of the eye are not well-maintained under static conditions18,19 and therefore study of isolated components of the eye require flow systems that can both maintain and assess metabolic processes. The fluidics system was developed to generate OCR and LPR data from a wide range of tissue types and in this paper we presented detailed protocols that were found to produce optimal results.
The major determinant for generating robust data using the flow system includes pre-equilibration of CO2-based media/buffer at 39 &#176;C (to ensure perifusate is not supersaturated with dissolved gas that would degas during the experiment). In particular, media or KRB buffer stored at 4&#160;&#176;C will be supersaturated relative to 37 &#176;C and will degas during the experiment if pre-equilibration times are insufficient. In addition, tissue loaded into the tissue chambers must not be traumatized by improper isolation of tissue due to tearing or incomplete separation of tissue, or by exposing tissue in low amount of bicarbonate-based buffer to atmospheric air for too long. The temperature control, flow stability and reliability of O2 detection have little variability and these factors do not contribute significantly to failure rate.
The instrument has eight flow channels/tissue chambers that run simultaneously which are supplied with perifusate from two reservoirs, four tissue chambers for each reservoir. To get the most accurate time-courses of OCR, kinetic curves are baseline corrected by chambers that are not loaded with tissue. Thus, a typical experimental protocol would involve two groups of three tissue chambers. Protocols in general fall into two categories: one is the different test compound protocols on each side (for instance drug/vehicle on one side of the MRM, and just vehicle on the other); the second is same test compound injection protocol on both sides of the MRM, but different tissue or tissue model on each side of the MRM. In this paper, the effects of oligomycin and FCCP on retina were compared to OCR by tissue that were not exposed to any test compounds, and two tissues were concomitantly assessed under the same protocol and conditions to identify tissue-specific behavior. The latter was illustrated in this study by showing increased dynamic range of metabolic rate by RPE-choroid-sclera relative to retina in parallel in the same experiment. Other reports have described a wider range of study designs including measuring the effects varying O2 levels on OCR and LPR, and concentration dependencies of fuels, drugs, and toxins20,21. In addition, although we have limited the analysis of outflow fractions to the measurement of lactate and calculation of LPR, the information content of an experiment increases greatly if multiple compounds and classes of compounds in the outflow fractions are assayed such as hormones, neurotransmitters, cell signals, and metabolites that can exit the cells20,22,23.
The loading of isolated retina or RPE-choroid-sclera is straightforward, and once isolated these tissues are simply placed into the top of the tissue chambers with forceps and allowed to sink down to the frit. RPE cells cultured on filter inserts develop appropriate polarization and markers of RPE maturity after 4-8 weeks in culture. It is not feasible to remove the RPE for live cell analysis once attached to the transwell membrane, if RPE maturity and polarization are to be maintained24. The perifusion chamber can accommodate strips of the transwell membrane that are cut with a scalpel while submerged in buffer and rapidly inserted into the tissue chambers. Although cutting filter strips has been placed into a static system24, no other fluidics method to assess these important cell types are available. The responses of RPE cells were rapid and more dynamic than either the retina or the RPE-choroid-sclera, likely in part due to immediate access of both the apical and basal aspects of the RPE cells configured as a monolayer on the membrane insert.
Another factor in assuring data has the highest signal to noise is selecting the optimal ratio of tissue loaded into the perifusion chambers relative to the flow rate. Too little tissue relative to the flow rate results in a difference of dissolved O2 concentration between inflow and outflow that is very small and difficult to measure reliably. In contrast, if the flow is too slow, then the concentration of O2 becomes so low that the tissue is affected by hypoxia. Nonetheless, the gas pressure-driven liquid flow can be maintained at flow rates down to 5 mL/min requiring only small amounts of tissue for accurate OCR and LPR measurements. In the experiments shown here, about 20 mL/min/channel was used which was suitable for either one retina, two RPE-choroid-scleras, or 360,000 RPE cells. To minimize the system effects that delay and disperse the exposure of the tissue to the injected test compound, multiple sizes of the tissue chambers are supplied, so that the amount of tissue (and flow rate) is matched with the appropriate size of the chamber.
Data from the analyses shown in this paper were represented in two ways: absolute magnitude with respect to rate, or fractional changes relative to a steady-state or baseline. The focus was on illustration of measurement of responses to test compounds. However, the fluidics system is well-suited to assess and compare effects of tissue treatment prior to the perifusion analysis such as genetic modifications. Testing whether a treatment is different from control is most robust if the effects of the treatment on normalized responses of test compounds is analyzed. If the analysis requires absolute magnitudes, the statistical power of the analyses of specimens that are pretreated is maximized if their assessment and controls are carried out in the same perifusion experiment.
Except for the stirrer, all parts that come in contact with liquid are supplied by the manufacturer as consumables and have been sterilized. These parts should not be reused, as experiments will occasionally be lost due to incomplete cleaning and contaminated surfaces. The system at the outset of setup is sterile. However, media is added to the MRM, and tissue is loaded in the chambers under non-sterile conditions. We have measured OCR in the system that is assembled with parts that are sterile, but where the experiment itself is carried out under non-sterile conditions. It takes about 14 h for bacteria to accumulate to the point of having measurable OCR (unpublished results). If protocols are used that are less than 10 h or so, then accumulation of bacteria and any effects due to these will be negligible.
Many investigators use instruments that are designed to measure OCR under static incubation of a monolayer of cells with a relatively high throughput25,26. In contrast, the fluidics instrument we have tested and described in this paper maintains tissue by ensuring adequate O2 delivery which is critical for the greater diffusion distances that are present in tissue specimens. In addition, it is able to collect fractions allowing assessment of multiple parameters in parallel with OCR which greatly enhances the ability to study relationships between them. Finally, dissolved gas concentrations (such as O2 and CO2) can be controlled, increasing the duration of experiments with bicarbonate-based media and buffer, enabling the user to study the effects of O2. It should be pointed out, a limitation for both methodologies is the inability to study the washout of test compounds, a functionality that other perifusion systems have4,27,28. Another consideration when determining the optimal analysis modality is the fact that fluidics systems use more media and test compounds than static systems. The extra expense is minimized with the current fluidics systems though due to the low flow rates that the system can be used.
Overall, a detailed description of the protocols to perform experiments with a new flow/assessment instrument is described. Data generated with retina and RPE-choroid-sclera recapitulated previous results obtained with systems that are much more difficult to use (and not readily available). It was also demonstrated that the system can maintain and assess RPE cells attached to transwell membranes, a very important cellular model that has not previously been analyzed with flow systems due to the fragility of the cells. The main parts of the protocol consist of a 75 min setup time, followed by a 90 min equilibration period and the experimental protocol making it suitable for routine use by laboratories that do not specialize in the operation of fluidics systems. Although we focused on measuring the acute response of tissue to test compounds, the system is very suitable to comparing tissue from various sources such as animal models or cells models that have been genetically altered or undergone test treatments/conditions. In addition, the scope of assays that can be conducted on the outflow fractions are wide-ranging and include metabolites, cell signaling molecules and secreted hormones/neurotransmitters as well as multi-component analysis generated by mass spectrometry on the fractions as well as the tissue.

Perifusion systems have a long history in the life sciences. In particular, for the study of secretory function by islets, they have been used to characterize the kinetics of insulin secretion in response to secretagogues1. In addition to collecting outflow fractions for subsequent assay of hormones and metabolites, real-time sensors have been incorporated, predominantly for the detection of oxygen consumption2,3,4. Widespread efforts to better understand mechanisms mediating diseases of the eye have been limited by a lack of physiologically relevant methods to assess metabolic regulation and dysregulation of the various isolated components of the eye, including the retina, retinal pigment epithelial (RPE)-choroid-sclera and cultured RPE cells. Static systems designed for cultured cells have been adapted for tissue5, but tissue requires flow for adequate oxygenation. Flow systems have been successful at accurately and reproducibly measuring real-time responses in oxygen consumption rate (OCR) by the retina and RPE-choroid-sclera, and the tissues stay metabolically stable for more than 8 h allowing highly informative protocols involving multiple test compounds4,6,7,8,9. Nonetheless, the operation of fluidics systems has historically required a custom-made apparatus and trained technical staff in non-standardized methodologies. Such systems have not been adopted as the standard methodology in most laboratories. The BaroFuse is a newly developed fluidics system that does not rely on pumps, but rather on gas pressure to drive flow through multiple channels and tissue chambers (Figure 1). Each channel is continuously monitored for OCR, and the outflow is collected with a plate-based fraction collector for subsequent assay of contents. Importantly, the tissue perifusion chambers for the instrument are designed to accommodate tissues of various geometries and sizes.
The heart of the instrument is the fluidics system, where flow is driven from a sealed, pressurized reservoir through small inner diameter (ID) tubing (contributing the most significant flow resistance in the fluid circuit) up into the glass tissue chambers that house the tissue. Pressure to the media reservoir module (MRM) is supplied by low-pressure and high-pressure regulators connected to a gas cylinder containing a mixture of gases (typically 21% O2, 5% CO2, balance N2), and the reservoir is sealed from the top by the perifusion chamber module (PCM) that holds the tissue chamber assemblies (TCAs). Flow rate is controlled by the length and ID of the resistance tubes and the pressure setting of a low-pressure regulator. Outflow tubes connected to the top of tissue chambers deliver fluid to either a waste receptacle (that is continuously weighed for automatic determination of flow rate) or into wells of a 96-well plate controlled by the fraction collector. The O2 detection system measures the lifetime of an O2-sensitive dye painted on the inside of each of the glass tissue chambers downstream of the tissue. This information is then used to continuously calculate OCR. The entire fluidics system resides in a temperature-controlled enclosure and the gas tank, fraction collector and computer are the major components of the instrument (Figure 2A). Finally, software that runs the instrument serves to control its operation (including the preparation and timing of injected test&#160;compounds, flow measurement system, and fraction collector timing), as well as processing and graphing the OCR data and other supplemental measurements.
In this paper we describe the protocols for using the fluidics system to perifuse and assess OCR and lactate production rate (LPR) for various isolated components of the eye. LPR is a parameter reflecting glycolytic rate that is highly complementary to OCR, where the pair accounts for the two major branches of energy generation from carbohydrates in the cell10. As preparation of the tissue and loading it into the tissue chambers is best learned by watching the procedure, the video will help illustrate several of the critical steps that are performed during set up and operation that are not easily conveyed by text alone.
The description of the protocol is divided up into 8 sections that correspond to different phases of the experiment (Figure 2B): 1. pre-experimental preparation; 2. preparation/equilibration of the perifusate; 3. instrument set up; 4. tissue equilibration; 5. experimental protocol; 6. instrument break down; 7. data processing; and 8. assays of outflow fractions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BIOLOGICAL SAMPLES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Envigo Harlan (Indianapolis, IN)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2920L9795</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270G</td>
			<td></td>
		</tr>
		<tr>
			<td>KCN</td>
			<td>Sigma-Aldrich</td>
			<td>60178</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactate&#160;</td>
			<td>MilliporeSigma</td>
			<td>L6661</td>
			<td></td>
		</tr>
		<tr>
			<td>Oliigomycin A</td>
			<td>Sigma-Aldrich</td>
			<td>75351L9795</td>
			<td></td>
		</tr>
		<tr>
			<td>CELL CULTURE AND TISSUE HARVESTING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beuthanasia-D</td>
			<td>Schering-Plough Animal Health Corp., Union, NJ</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Euthasol, 390 mg/ml sodium pentobarbital</td>
			<td>Virbac</td>
			<td>RXEUTHASOL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>12303C</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Buffered Salt Solution</td>
			<td>GIBCO</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs Ringer Bicarbonate (KRB)</td>
			<td>Thermo Fisher Scientific</td>
			<td>J67795L9795</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>#CB-40230</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCKi&#160;</td>
			<td>Selleck Chemicals</td>
			<td>Y-27632</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher</td>
			<td>#25-200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPPLIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Cylinders: 21% O2/5% CO2/balance N2</td>
			<td>Praxair Distribution, Inc</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell filters&#160;</td>
			<td>MilliporeSigma</td>
			<td>3470</td>
			<td></td>
		</tr>
		<tr>
			<td>COMMERCIAL ASSAYS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red Glucose/Glucose Oxidase Assay Kit</td>
			<td>ThermoFisher</td>
			<td>A22189</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase from Aerococcus viridans</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>A22189L9795</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactate Oxidase</td>
			<td>Sigma-Aldrich</td>
			<td>L9795</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BaroFuse&#160; Multi-Channel Perifusion system</td>
			<td>EnTox Sciences, Inc (Mercer Island, WA</td>
			<td>Model 001-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 4 Fluorometer</td>
			<td>BioTek (Winooski, VT)</td>
			<td>S4MLFPTA</td>
			<td></td>
		</tr>
	</tbody>
</table>

maintaining and assessing various tissue and cell types of the eye using a novel pumpless fluidics system

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

To illustrate the resolution of the data generated from isolated components of the eye, OCR and LPR was measured with three types of tissue (retina, RPE-choroid-sclera, and RPE cells) following a commonly used protocol (the mitochondrial stress test10; Figure 10, Figure 11, and Figure 12). The amount of tissue used for each tissue is shown in Table 1. Data was processed and graphed using the software package that was developed for the fluidics system. The preparation of retina and RPE-choroid-sclera is relatively straight-forward and takes less than 20 min for each tissue type. OCR was constant during the time that test compounds were injected, indicating stable health and function of the tissue and supporting the validity of the method (Figure 10). Once validated for each tissue type, we have not found it necessary to run controls where no test compounds are injected for each experiment. Consistent with data obtained using more conventional perifusion methods6,8,13, OCR decrease in response to oligomycin and increased OCR in response to FCCP. Changes in LPR were in the opposite direction of those observed for OCR: oligomycin increased LPR, which then decreased (but only slightly) in response to FCCP (Figure 11). To compare the statistical significance of the effect of each sequential test compound, t-tests were performed (which are calculated automatically by the software that comes with the instrument). Since the goal of the paper was to describe how to perform the method, the number of replicates carried was not always high enough to produce statistical significance. In general, though, when the number of replicates were 3 or more, effects of FCCP and oligomycin on both OCR and LPR were significant.
RPE cells have not been previously analyzed with flow systems but responded similarly to RPE-choroid-sclera (consistent with the view that a large fraction of OCR is due to RPE cells; Figure 11). These illustrative examples highlight ability of the system to maintain tissue viability as reflected by the stability of OCR in the control channels, and the high signal to noise ratio for changes in OCR of the magnitude induced by oligomycin and FCCP, which was more than 100 to 1. In addition, assays of outflow fractions can be used to correlate the rate of uptake or production of a wide array of compounds exchanging with the extracellular fluid are complementary to OCR (in this case, LPR). These features of the instrument allowed accurate quantification of characteristic differences in tissue responses between tissue types performed in parallel. OCR by RPE-choroid-sclera and RPE cells are consistently more sensitive to oligomycin than retina (Figure 11 and Figure 12), although for the RPE-choroid-sclera the duration of exposure to FCCP was not long enough to reach steady state. A point to consider arose when using DMSO as a solvent. At higher concentrations, (0.2%) DMSO had a transient effect on OCR by retina (presumably reflecting an effect of a change in osmotic pressure brought about by DMSO&#39;s effect on membrane permeability).
Based on the assumption that KCN completely inhibits respiration by its direct action on cytochrome c oxidase, OCR at the end of the KCN exposure is set to 0 and all OCR values are calculated based on the change relative to the KCN value. OCR can occur independent of the respiratory chain and cytochrome c oxidase. However, the magnitude of this contribution to overall OCR is generally not more than a few percent (data not shown) and the extended length of time that tissue is exposed to KCN ensures that substrates of oxidases that are not part of the electron transport chain have been depleted.
Statistical analysis 
Single experiments were shown as indicated in the figures, but with multiple channels that were averaged. Data was then graphed as the average &#177; the standard error (SE; calculated as SD/&#8730;n).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65399/65399fig1v2.jpg" /> 
Figure 1.&#160;Schematic of the fluidics/assessment system. Major components include the enclosure, temperature control elements, fluidics and tissue chamber systems, regulation of gas pressure in the head space above perifusate, fraction collector/flow rate monitoring, and O2 detectors. Abbreviations: MRM = Media Reservoir Module, PCM = Perifusion Chamber Module, TCA= Tissue Chamber Assemblies. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65399/65399fig02.jpg" /> 
Figure 2. (A)&#160;Picture of the major components of the instrument. The major components consist of gas tank (pressure regulators), enclosure, fraction collector and computer. (B)&#160;Experimental flow chart showing the major categories of steps and the time it takes to complete them. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65399/65399fig03.jpg" /> 
Figure 3. View of the MRM. The MRM is shown with an MRM insert (left) and stir bars (right) placed into the bottom of the MRM inserts (placed in each side of the MRM Divider). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65399/65399fig04.jpg" /> 
Figure 4. Tubing assembly and purge tubing assembly in the MRM. (A)&#160;Test compound injection tubing assembly and purge tubing assembly attached to ports on the MRM. (B-C) The test compound injection assembly and purge tubing assembly (B) are placed in the groove in&#160;the front of Enclosure (C). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65399/65399fig05.jpg" /> 
Figure 5. Powering up the MRM temperature controller. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65399/65399fig06.jpg" /> 
Figure 6. Tissue chambers and gas tank.&#160;Positioning the O2 detector on the detector stand (which also supports the MRM and PCM), and placement of the band around the fins of the PCM that help secure the tissue chambers in place. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65399/65399fig07.jpg" /> 
Figure 7. (A) High- and low-pressure regulators on the gas tank. (B-C) Purge tube. Purge tube allows the headspace in the MRM to clear of air and fill with gas from the supply tank. Pictures showing open purge tube (B) and close purge tube (C). Test compound injection assembly stays closed through the purge process. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65399/65399fig08v3.jpg" /> 
Figure 8.&#160;Tissue chamber and the outflow setup. (A) Dimensions of the Transwell membrane after it is cut into three strips of equal width. (B) Outflow multi-tube support. (C) Outflow multi-tube support positioned on the lip of the enclosure with the tubing adapters near the tissue chambers. (D) Picture of outflow tubing assemblies attached to the tissue chambers. (E)&#160;Aerial view of the enclosure without the Lid. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig08largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65399/65399fig09.jpg" /> 
Figure 9. Injection of compound in MRM. Injecting a test compound through the injection port into the MRM using a 5 mL syringe. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65399/65399fig10.jpg" /> 
Figure 10.&#160;OCR and LPR curves in response to test compounds. OCR and LPR by retina isolated from mice (1 retina/channel) in response to the presence or absence (control) of test compounds as indicated. Each curve is the average of 6 replicates from a single experiment (error bars are SE; p-values are calculated by performing paired t-tests comparing steady state values for each test agent with that of the previous test agent). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65399/65399fig11.jpg" /> 
Figure 11.&#160;OCR curves. OCR by RPE-choroid-sclera and retina isolated from mice (1 retina or 2 RPE-choroid-sclera/channel) measured in parallel in response to test compounds as indicated. Data is the average of replicates from a single experiment (n = 2 and 4 for RPE-choroid-sclera and retina respectively; p-values are calculated by performing paired t-tests comparing steady state values for each test agent with that of the previous test agent). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65399/65399fig12.jpg" /> 
Figure 12.&#160;OCR and LPR curves from RPE cells. OCR and LPR from RPE cells attached to transwell membranes that were cut into strips and loaded into the perifusion chambers. Data is the average of replicates from a single experiment (n = 3, with 1.5 membranes/channel (360,000 cells/channel); p-values are calculated by performing paired t-tests comparing steady state values for each test agent with that of the previous test agent). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>TISSUE/CELL</td>
			<td>Amount/Channel</td>
			<td>FLOW RATE: mL/min</td>
		</tr>
		<tr>
			<td>Retina (mouse)</td>
			<td>1</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>RPE-choroid-sclera (mouse)</td>
			<td>2</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>RPE Cells on Transwell Membranes</td>
			<td>360,000 Cells (4 x 1/3 filter strips)</td>
			<td>0.016</td>
		</tr>
	</tbody>
</table>
Table 1. Recommended operating specifications for different tissue.
Supplementary Figure 1. Graphical representation of experimental design. Timing and composition of exposure to test compounds, and timing of fraction collection. Concentration increment (Conc Inc) is the change in concentration to be implemented. <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2. User interface at startup. UI of the startup window of the O2 detection software that monitors the O2 in the tissue chambers inserted into the PCM. <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3. User interface for experiment settings. UI for entering experimental information (left) and selecting times for collection of outflow fractions (right). <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig3.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4. User interface of the injection page. Injection page which calculates injection volumes based on desired concentrations of test compound and volume left in the MRM. <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig4.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Methods for tissue sample preparation. <a href="https://www.jove.com/files/ftp_upload/65399/supplementary file 1_RE.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment at JoVE.com

Author Spotlight: Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell ...

maintaining-assessing-various-tissue-cell-types-eye-using-novel

Research

JoVE Journal

Bioengineering

1.1K Views.  EnTox Sciences, Inc. Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models. 

Video: Author Spotlight: Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment 

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...