xcelligence cardio system project proposal: contractile...

Xcelligence Cardio System Project Proposal: Contractile

Functions of Gene Targeted CardiomyocytesPhase I: Optimization of isolation, plating and culturing of neonatal mouse cardiomyocytes (NMCs)

Phase 1 of the project investigated the optimization of isolation procedures, plating and culture conditions of neonatal mouse cardiomyocytes for use in the Roche/Acea Xcelligence Cardio System.

1. Isolation Procedure

Isolation of cells was performed using previously published protocols (adapted from Perriard, ETH Zurich, Switzerland; Gautel and Ehler, Kingʼs College London, UK; see also References below) with optimization in enzyme mixture and media formulation.

Media

Isolation medium Digestion medium

20mM BDM (Sigma B-0753)0.0125% Trypsin (cellgro 25-053-Cl)HBSS (without Ca2+, Mg2+)

20mM BDM2mg/ml Roche Collagenase/Dispase enzyme mix (10269638001)L-15 medium

Plating medium Maintenance medium

65% DMEM High Glucose16% M-19910% Horse Serum5% Fetal Calf Serum1% Penicillin/Streptomycin (cellgro 30-002-Cl)

78% DMEM High Glucose17% M-1994% Horse Serum1% Penicillin/Streptomyzin (cellgro 30-002-Cl)1µM AraC (Sigma C6645)

S. Lange, PhD. UCSD; Roche Xcelligence cardio system project proposal - PHASE 1 results! /8

Isolation of cellsThe following procedure is a two day protocol for the isolation and culture of neonatal mouse cardiomyocytes. All solutions are sterile or sterile filtered. All tools are sterilized by surface sterilization with 75% EtOH. Except for the initial tissue extraction, all steps are performed in sterile laminar flow cell culture hoods. This protocol is intended for the isolation of neonatal mouse hearts from one litter - approximately 5-12 pups. Scale media/enzyme usage as appropriate.

Day 1Prepare 50ml of 1xPBS (without Ca2+, Mg2+) supplemented with 20mM BDM and disperse into two bacterial dishes on ice. Prepare 10ml of isolation medium in 50ml conical Falcon tube. Keep all solutions on ice. Sterilize scissors (one curved, one straight), forceps (curved, Dumont No.7).

• Neonatal mice (recommended: 1-3 days old) are rinsed quickly in 75% EtOH solution for surface sterilization. Pups are decapitated using sterile scissors (straight), and chests are opened along sternum to allow access to chest cavity and hearts. Hearts are extracted from body with curved scissors and transferred immediately into bacterial dish containing 1xPBS (without Ca2+, Mg2+) with 20mM BDM on ice. All following steps are performed in the sterile cell-culture hood.

• Remove lung tissue (and atria if desired). Wash hearts to remove blood. Remove washed hearts from first dish and transfer into second bacterial dish containing 1xPBS (without Ca2+, Mg2+) with 20mM BDM (on ice) using forceps. Transfer cleaned/washed hearts into a drop of isolation medium in a third bacterial dish (on ice) and use curved scissors to mince hearts into small pieces (approximately 1mm3, or smaller). Transfer minced hearts into conical tube containing 10ml of the isolation medium (on ice), and incubate with gentle agitation at 4ºC over night.


Day 2Prepare 10 ml digestion medium, 30ml L-15 medium supplemented with 20mM BDM and 10 ml plating medium. Coat cell-culture plates with collagen solution (Sigma C-8919). Dry collagen coated cell-culture dishes in sterile laminar flow cell-culture hood.

• Remove conical tube containing predigested hearts from 4ºC. Tissue fragments should be aggregated. Let the tissue fragments sink to the bottom of the tube and remove the supernatant (make sure not to loose any tissue). Add 5ml of digestion medium and 5 ml of L-15 supplemented with 20mM BDM to tissue fragments and oxygenate suspension using oxygen or air for 1 minute. Transfer sealed conical tube containing hearts in digestion medium to 37ºC water bath for 2 minutes. Incubate hearts at 37ºC with gentle agitation for 20-30 minutes. Caution: longer incubation periods may reduce cell viability.

• Prepare cell-strainer (40-100µm nylon mesh; e.g. BD Falcon 352340) and place in fresh sterile 50ml conical falcon tube. Pre-wet cell strainer with 5ml L-15 supplemented with 20mM BDM. Triturate tissue fragments using a pre-wetted 10ml cell-culture pipette for about 10-20 times. The tissue fragments should mostly disperse during this step, releasing the cells into suspension. Let larger tissue fragments sediment and transfer supernatant containing suspended cells into fresh conical tube through cell-strainer. Re-suspend undigested tissue fragments in 5ml digestion medium and incubate for additional 5-10 minutes at 37ºC with gentle agitation.

• After incubation triturate remaining tissue and add to conical tube containing cells from the first digest through cell strainer. Rinse cell-strainer with 10ml L-15 supplemented with 20mM BDM to allow passage of all digested cells. Centrifugate conical tube containing suspended cardiomyocytes for 5 minutes at 3000rpm (50-100g). Remove supernatant (containing mostly fibroblasts and endothelia cells) and re-suspend cell pellet in 10ml plating medium.

• Plate cells into 10cm cell culture dish and incubate cells for 2-3 hours in cell culture incubator. This pre-plating step removes fibroblasts and endothelia cells, which will adhere to the uncoated cell-culture dish.

• After incubation, wash non-adherent cardiomyocytes from 10cm culture dish (re-suspend cells by repeatedly pipetting suspension over the dish) and transfer to sterile 15ml conical Falcon tube. Optional: If necessary repeat pre-plating step to remove more fibroblasts from cell-suspension.


• Count cells (e.g. by using Neubauer hemocytometer). Plate cells into collagen coated E-plates with a density of approximately 8*104-1*105 cells per well (or approximately 3*105 cells per cm2; see below “2. Optimization of plating and culture conditions”). Place dish into cell culture incubator and culture undisturbed for 12-18 hours.

Day 3 - onward

• One day after plating cardiomyocytes should have adhered to the cell culture dish and optimally start spontaneously to contract. Replace plating medium with maintenance medium (containing AraC to inhibit fibroblast proliferation, see below “2. Optimization of plating and culture conditions”), and culture for additional 1-5 days before assay. Change medium as necessary.

References

• Worthington Tissue Dissection Guide, Worthington Biochemical Corp. Freehold, NJ, 1990.

• Worthington Enzyme Manual. Freehold, NJ, 1993.

• DeRobertis EDP, Saez FA and DeRobertis EMF. Cell Biology. 6th Ed. WB Saunders Co. Philadelphia, PA, 1975

• Engelmann GL, McTiernan C, Gerrity RG, Samarel AM. Technique 2, 279, 1990

• Freshney RI. Culture of Animal Cells - A Manual of Basic Technique, 4th Ed. Wiley-Liss Inc. New York. 2000.

• Gross WO, Schopf-Ebner E, and Bucher OM. Technique for the Preparation of Homogenous Cultures of Isolated Heart Muscle Cells. Exp Cell Res. 53(1). 1968.

• Hanks, JH and Wallace RE. Proc Soc Exp Biol Med. 71(169). 1949

• Jakoby WB, and Pastan IH. Methods Enzymol LVIII. 121. 1988

• Leibovitz A. Am J Hyg 78(173).1963

• Speicher DW, and McCarl RL. Pancreatic Enzyme Requirements for the Dissociation of Rat Hearts for Culture. In Vitro. 10(30). 1974

• Toraason M, Luken ME, Breitenstein M, Krueger JA, and Biagini RE. Comparative Toxicity of Allylamine and Acrolein in Cultured Myocytes and Fibroblasts from Neonatal Rat Heart. Toxicology. 56(107).1989.


2. Optimization of plating and culture conditions

Optimization of plating and culture conditions included these variables:1. Pre-plating to remove fibroblast from cell population2. Coating of dishes with extracellular matrix components3. Plating density4. Inhibition of cardiac fibroblast proliferation5. Addition of chronotropic agents to induce sarcomerogenesis and spontaneous

cardiomyocyte contraction.

Pre-platingPre-plating of cells after isolation greatly removes cardiac fibroblasts and endothelia cells from the whole cell mixture. A single pre-plating step removes approximately 50-80% of non-cardiomyocytes from the cell-population. Repetition of pre-plating more than twice, as well as of pre-plating times longer than 3 hours is not recommended, due to additional loss of cardiac myocytes. Substitution of pre-plating with ficoll-gradient (or cell-sorting) may improve purity of cardiomyocyte cell population (not tested). Lack of pre-plating will result in cardiac fibroblasts preferentially adhering to inter-electrode space, whereas the electrode is populated by cardiomyocytes (see Figure 2A). This may result in incomplete cardiomyocyte cell communication and potentially skewed results in the Xcelligence cardio-plate assay.

CoatingCoating of cell-culture dishes/E-plates with extracellular matrix (ECM) components like collagen, fibronectin or laminin positively influences cardiomyocyte adherence and cell spreading during the initial plating step and for subsequent culture period. Coating of dishes also greatly reduced the preferential adherence of cardiomyocytes to the electrode materials, vs. the material of the glass wafer. Moreover the cell monolayer on coated dishes appeared more homogenous. We tested coating of E-plates with a 0.1% collagen solution (Sigma C-8919), or a 1:1 combination of 0.1% collagen and 1% gelatin (in 0.1N acetic acid, sterile filtered). E-plates were incubated with the ECM solutions for 1 hour, and after removal of ECM dried in the laminar flow cell-culture hood.


Plating densityWe analyzed optimal cell concentrations for plating of neonatal cardiomyocytes on E-plates. Cardiomyocyte cultures were analyzed by immunofluorescence microscopy for confluency, homogeneity and formation of cell-aggregates.Optimal cell concentrations for plating should result in a near-confluent cardiomyocyte monolayer to achieve good results during subsequent contractility analysis in the XCelligence system. Plating of cardiomyocytes in too low concentrations results in preferential adherence of cells on electrodes, but not the glass-wafer material (inter-electrode space), whereas high concentrations result in multilayered cardiomyocyte aggregates and low homogeneity.

Figure 1. Effect of cell-density on plating conditions.

density too low optimal density density too high• cardiomyocytes adhere

preferentially to electrode material• inter-electrode space void of cells• cells are not confluent, cell-cell

connections only established along electrode, but not between electrodes.

• cells form monolayers with near-confluency

• cell-cell contacts established between cells spanning the inter-electrode space.

• multi-layered cardiomyocytes: cardiomyocytes form aggregates (white arrows)

• low homogeneity of cell layers results in areas void of cells (black arrowhead).


Addition of proliferation inhibitorsSince differentiated cardiomyocytes do not undergo cell-division, the addition of proliferation inhibitors, such as 10µM AraC (Cytosine-B-D-arabino-furanoside hydrochloride; Sigma C6645) to the maintenance medium is highly recommended. Even with the addition of pre-plating steps to reduce the population of cardiac fibroblast and endothelia cells, the remaining fibroblasts will undergo cell proliferation and significantly change the cell-population on the E-plate over time. Moreover, cardiac fibroblasts may preferentially settle between electrodes and act as insulators (inhibit cardiomyocyte communication/electrical coupling).

Figure 2. Effects of pre-plating, and inhibition of fibroblast proliferation using AraC.A) Culturing of either complete isolated cell suspension without prior pre-plating step (see “1. Isolation Procedure”), or without the supplementation of AraC to the maintenance medium results in in-homogenous cell populations, with cardiomyocytes preferentially localized to electrodes, and cardiac fibroblasts adhering to inter-electrode space (white arrowhead).B) Addition of pre-plating step during isolation procedure to remove cardiac fibroblasts or endothelia cells from cell mixture, and supplement of AraC to maintenance medium results optimally in homogenous mono-layer of cardiomyocytes on electrode and inter-electrode space. DAPI - blue in overlay; filamentous actin (F-Actin, green in overlay; top-right panel in A, B) labels all cells, whereas sarcomeric marker alpha-Actinin (red in overlay; bottom-right panel in A, B) marks only cardiomyocytes.


Addition of chronotropic agents.Supplementation of the maintenance medium with agents, such as phenylephrine (0.1mM, preferentially for neonatal rat cardiomyocytes; Sigma P6126), or isoproterenol (1µM, preferentially for neonatal mouse cardiomyocytes; Sigma I6501) greatly increases sarcomerogenesis, spreading of cardiomyocytes on the plate as well as spontaneous beating of cells. However, the addition of these agents should be weighed with respect to desired assay parameters (e.g. addition may skew contractile behavior and mask or induce arrhythmias).


xcelligence cardio system project proposal: contractile...

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