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Journal of Biotechnology 184 (2014) 146–153

Contents lists available at ScienceDirect

Journal of Biotechnology

j ourna l ho me pa ge: www.elsev ier .com/ locate / jb io tec

icroscale acoustic disruption of mammalian cells for intracellularroduct release

ay Ly, Franklin Lu1, Gargi Maheshwari, Shyamsundar Subramanian ∗

accine R&D, Merck & Co., Inc., West Point, PA 19486, USA

r t i c l e i n f o

rticle history:eceived 30 October 2013eceived in revised form 21 March 2014ccepted 28 April 2014

vailable online 23 May 2014

. Introduction

The development of manufacturing processes for mammalianell derived biopharmaceuticals increasingly relies on high-hroughput (HT) approaches using scale-down models for selectinglones during cell line development, optimizing the cell culturerocess during development, and troubleshooting manufacturing

ssues (Bareither and Pollard, 2011; Bhambure et al., 2011; Lai et al.,013). High-throughput screening (HTS) using mammalian cells islso used heavily in drug discovery compound screening (Mayrnd Bojanic, 2009; Wunder et al., 2008) and biological investiga-ions requiring HT gene-knock down (RNAi) or over-expressionDemir and Boutros, 2012; Skalamera et al., 2011). Significantffort has thus been focused on developing HT systems to cul-ivate and analyze mammalian cells at microscale for cell-basedcreening applications (Amanullah et al., 2010; An and Tolliday,010; Astashkina et al., 2012; Bareither and Pollard, 2011; Kim et al.,012; Michelini et al., 2010).

As a result, numerous solutions exist for non-destructivelyeasuring the cells (directly or indirectly) and secreted products

recombinant proteins, budded viruses, metabolites) that serves readouts in HT cultivation systems. A variety of intracellularr cell-associated components (receptors, cytoskeletal proteins)an be measured using microscopy or flow cytometry methodsithout the need for extraction. However, some viruses, virus-

ike particles (VLPs), membrane proteins, and cytoplasmic enzymes

ust be extracted and remain functional after processing. These

ntracellular targets and their associated analytical methods canuffer detrimental effects due to the method of cell disruption. For

∗ Corresponding author: Tel.: +1 215 264 4872.E-mail address: shyamsundar.subramanian@gmail.com (S. Subramanian).

1 Current address: Stanford University, CA, USA.

ttp://dx.doi.org/10.1016/j.jbiotec.2014.04.030168-1656/© 2014 Published by Elsevier B.V.

instance, ionic and non-ionic detergents are not compatible withenveloped viruses or enveloped VLPs that have a lipid membrane.Individual membrane proteins extracted from lipid membranesusing detergents can also lose activity (Seddon et al., 2004; Popot,2010). Even for products that are not as detergent sensitive (e.g.non-enveloped viruses or VLPs), the presence of residual deter-gent may interfere with subsequent cell-based assays (infectivityassays). The presence of detergents even interferes with com-monly used analytical methods for measuring protein and DNAconcentration (Bradford, 1976; Lowry et al., 1951; Singer et al.,1997).

To address these challenges with detergent use, we sought todevelop a HT (96-well plate format) mechanical method for dis-rupting adherent mammalian cells and releasing an intracellularproduct for screening. Several instruments capable of providingmechanical lysis using sonic energy in a 96-well format wereconsidered, such as the Covaris E210 (Covaris, Inc.), SonicMan(Brooks Life Science Systems) and Hielscher UIP250MTP (HielscherUltrasonics GmbH). These instruments have been used for manyapplications, such as compound dissolution (Oldenburg et al.,2005), virus extraction (Nauwelaers et al., 2009), and cell/tissuedisruption to extract metabolites or proteins (Kassama et al.,2010; Toorchi et al., 2008). The Covaris adaptive focused acous-tics (AFA) technology for controlled ultrasonication lyses samplesusing focused acoustic waves (Laugharn and Garrison, 2004) andhas previously been used as a scale-down method for biopharma-ceutical product recovery from microbial systems. Wenger et al.(2008) applied this instrument for disruption of yeast cells torecover human papilloma virus VLPs and, more recently, Li et al.(2012) evaluated this technology for the recovery of antibody frag-

ments from Escherichia coli. In contrast to these uses of AFA todisrupt 2–8 mL cultures of microbial cells with high energy deliv-ery, a gentler disruption method for releasing a labile productfrom mammalian cells at microscale (∼100 �L) was desired. To

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ur knowledge, there is currently no reported method for achiev-ng the goal of HT microscale mechanical lysis of mammalianells for this purpose. As a representative system, we chose aighly cell-associated virus (Varicella-Zoster Virus) propagated

n mammalian cells (MRC-5) that is the basis for marketed vac-ines against Chicken Pox and Shingles (Brunell, 1967; Schmidtnd Lennette, 1976). The current procedure for determining virusnfectivity in this system requires tip-horn sonication for recov-ry and cannot be operated at a scale less than 10 mL (Singhvit al., 1996). A method to measure infectious virus in 96-welllate cultures could enable substantial HT process developmentor vaccines produced using mammalian cell systems like this one.n addition to viral vaccine applications, this microscale disrup-ion method may provide an alternative to detergent-based lysisor the release of other intracellular components in HTS applica-ions.

. Materials and methods

.1. Cell culture in 96-well plates

MRC-5 cells (American Type Culture Collection, Manassas, VA)ere cultivated in tissue culture treated flat bottom 96-well plates

BD Biosciences, Bedford, MA) at 37.0 ◦C in a 5% CO2 humidifiedncubator using Williams’ Modified E Medium (WMEM, HyClone,ogan, UT) supplemented with �-irradiated, iron-supplemented,ovine calf serum (BCS, HyClone, Logan, UT). Each well used 200 �Lf growth media. For cell cycle analysis, MRC-5 cells were planted atarious cell densities (0.1–1.6 × 105 cells/cm2) cultured for variableuration post-plant before harvesting. For virus infection, MRC-5ells were inoculated at a plant density of 1.6 × 105 cells/cm2 andncubated for approximately 24 h prior to virus infection. Excep-ions to these standard conditions are noted in individual figureegends.

.2. Trypsinization of 96-well plates

The adherent cell layer in harvested 96-well plates was washedwo times with 100 �L Dulbecco’s phosphate buffered saline (DPBS;AFC, St. Louis, MO) while being careful not to disturb the cell layeruring buffer addition and aspiration. Cells were then incubatedith 40 �L trypsin-citrate (0.25%; SAFC) at 37.0 ◦C (uninfectedlates) or 35.5 ◦C (virus infected plates) for 8 min. Plates were vor-exed briefly and trypsin activity was quenched with 160 �L ofrowth media per well. Cells were dispersed in the medium throughepeated pipetting. Cell concentration in trypsinized cell suspen-ions was measured using the Guava ViaCount reagent and protocoln the Guava EasyCyte flow cytometer (EMD Millipore, Billerica,A).

.3. Cell cycle analysis

96-well plates were trypsinized as described above and cen-rifuged at 450 × g at ambient temperature for 5 min to pellet cells.ollowing centrifugation, the supernatant was discarded and cellsesuspended in 200 �L DPBS. The centrifugation and aspiration ofupernatant was repeated a second time. Cells were then resus-ended in residual DPBS and fixed by adding 200 �L of 70% ice-coldthanol (Thermo Fisher Scientific, Waltham, MA) in a drop-wiseanner while agitating the plate on a shaker platform. The 96-well

lates were then sealed and incubated at 4 ◦C for at least 1 h (up

o 12 h) to enable fixation to occur. Fixed samples from duplicateells were pooled and centrifuged to pellet cells, and the ethanol in

he supernatant was discarded by aspiration. The Guava cell cycleeagent (containing propidium iodide; EMD Millipore) was then

logy 184 (2014) 146–153 147

added to each sample (200 �L/well) and incubated in a round bot-tom low cluster plate (Corning, Lowell, MA) for 30 min on ice, andprotected from light. Cell cycle data was acquired using the Guavacell cycle protocol on a Guava EasyCyte system. Cell cycle data wereanalyzed by first gating a dot plot (forward scatter vs. DNA content,PM2) to remove debris. Gaussian fits of the DNA histogram (area)were used to quantify the fraction of cells (%) in the G0/G1, S andG2/M cell cycle phases (FCS Express version 3 software, De NovoSoftware, Los Angeles, CA).

2.4. Virus infection in 96-well plates

MRC-5 cells in 96-well plates were infected with attenuated VZV(vOka/Merck VZV strain; Singhvi et al., 1996) at various multiplic-ity of infection (MOI) ranging from 1:4 to 1:128 plaque formingunit/viable cell (PFU/cell). The VZV infected MRC-5 cells were incu-bated for at least 12 h at 35.5 ◦C in a 5% CO2 humidified incubator.See figure legends for specific plant cell densities, MOI, and harvesttimes used in each study.

2.5. Harvest of 96-well plates for sonication

96-well plates were harvested at various times post-plant ofcells or post-infection with virus. Media was aspirated and replacedwith 100 �L virus stabilizer, and plates were stored at −70 ◦C untiltreatment with the sonication instrument.

2.6. Cell lysis by sonication using Covaris E210

The instrument is comprised of a transducer, power supply,recirculating water bath and sample plate holder. The transducerdelivers energy in the form of acoustic waves to the sample focalzone and the water bath acts as a coupling medium for energy trans-fer (Fig. 1). An intensifier (Part No.500141, Covaris, Inc., Woburn,MA) was placed directly above the transducer to increase processefficiency. The Covaris SonoLABTM application software was usedto control the instrument. The power delivered to the sample wasvaried by adjusting the intensity, duty cycle, and treatment timeto optimize cell lysis. The intensity represents the acoustic waveamplitude, which is proportional to the energy in millivolts. Theintensity ranges from 0.1 to 10. Duty cycle represents the frac-tion of time that the transducer is actively generating acousticwaves and ranges from 0.1 to 20%. The adjustable Z-offset rep-resents the vertical positioning of the plate from the transducerto direct the point where the energy converges inside the samplecontainer (Laugharn and Garrison, 2004). Cycles per burst (cpb)refers to the number of acoustic waves generated by the trans-ducer during the duty cycle and ranges from 50 to 100. The Z-offsetand cycles per burst were kept constant at −3 mm and 100 cpb,respectively. Frequency sweeping was used for all AFA treatment.Frequency sweeping applies a range of frequencies during sampletreatment (Laugharn and Garrison, 2004).

Prior to sample treatment, the 96-well plates containing 100 �Lsample per well were thawed in a 30 ◦C water bath for 15 min. Theplate was sealed using a sterile, ethylene vinyl acetate sealing mat(Thermo Fisher Scientific) to prevent cross-contamination of sam-ple due to splashing from the AFA treatment. The sample plate wasplaced on a plate holder, which was positioned above the trans-ducer. Sonication was performed in an isothermal bath to maintainsample temperature (7–9 ◦C). Each well was treated individuallyand sequentially. The intensity, duty cycle and treatment time wereset at the desired settings using the Covaris SonoLABTM applica-

tion software. After AFA treatment, the samples were centrifugedat 430 × g at 4 ◦C for 1 min to recover any sample that may havesplashed up onto the inner surface of the sealing mat. The sampleswere frozen at −70 ◦C and stored until use in the virus infectivity

148 M. Ly et al. / Journal of Biotechnology 184 (2014) 146–153

Fig. 1. Schematic diagram of the Covaris E210 Adaptive Focused Acoustics Instrument. The sealed 96-well plate containing sample sits on the plate holder which is submergedi ansdua ide thw

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ield assay. Sample treatment was non-invasive and took approx-mately 30 min for an entire 96-well plate (at 20 s per well).

.7. Cell lysis by sonication using SonicMan or HielscherIP250MTP

The SonicMan unit (Brooks Life Science Systems, Spokane, WA)onsisted of a power supply that delivered acoustic energy through

sonic horn that was placed directly above the 96-pin lid. Themount of energy delivered to the sample was varied by adjus-ing the power setting (1–100%), treatment time, and number ofonication cycles on the software that was included as part of theain unit. The SonicMan did not have an option for a water bath to

ontrol sample temperature. Bio-burden on the non-sterile pin lidas reduced by decontamination with 70% ethanol solution prior

o placement of the pins into the 96-well plate containing the sam-le. The SonicMan 96-pins were submerged in each of the wellsontaining the harvested cells treated at a power setting of 30% forumulative sonication times of 10–90 s.

The Hielscher UIP250MTP (Hielscher Ultrasonics GmbH, Tel-ow, Germany) included a power supply, transducer and sonotrodeith reservoir. The 96-well plate was placed in the water-filled

eservoir at the top of the sonotrode and the power was suppliedia the transducer, through the sonotrode to the bottom of thelate. The water supply for the reservoir came from a recirculatingater bath that enabled sample temperature control. The amount

f energy applied to the sample was varied by adjusting the ampli-ude ranging from 20 to 100%. Samples were sonicated for 30 s atn amplitude setting of 100%. Both the SonicMan and HielscherIP250MTP sonicated all 96 wells in a plate concurrently.

.8. Visualization of cell lysis and particle size analysis

Light microscopy was used to observe MRC-5 lysis after AFAr sonication treatment. Images of samples were taken using thelympus IX50 light microscope (Olympus America Inc., Centeralley, PA) and SPOT Advanced software version 4.0.8 (Diag-ostic Instruments, Inc., Sterling Heights, MI). The CASY® 1 Cellounter + Analyzer System (Innovatis AG, Reutlingen, Germany)

as used for particle counting. A 150 �m CASY® capillary was used

o measure particles in the range of 3.3–20 �m. A volume of 80 �Lf AFA treated sample was diluted 62.5 times with CASY®ton, ansotonic diluent solution. The diameter of trypsinized MRC-5 cells

cer. Acoustic energy is transferred from the transducer through the intensifier platee well of a 96-well plate. AFA settings and automatic maneuver of the 96-well plate

was measured using Vi-CELL® XR (Beckman-Coulter, Indianapolis,IN).

2.9. Virus infectivity yield assay

Sonicated 96-well plates were thawed in a 30 ◦C water bath for15 min. The semi-automated flow cytometric VZV infectivity assaydeveloped by Gates et al. (2009) was modified and used to deter-mine relative virus infectivity yield of samples treated with theCovaris AFA in a 96-well plate system without a reference standard.The sonicated sample (80 �L) was used to infect the MRC-5 cellculture plates to test for infectivity with a neat sample and dilu-tions of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. After approximately96–120 h of incubation after infection, the plates were trypsinizedas described above and quenched using 60 �L of working buffer(DPBS supplemented with 5%, v/v FBS) containing the primary andsecondary antibodies. The primary antibody against VZV glycopro-tein H (anti-gH; Clone A1; IgG2a; Merck & Co., Inc., West Point, PA)and the secondary antibody, Alexa Fluor® 488 F(ab)’2 Goat Anti-Mouse IgG (H + L) (Life Technologies, Grand Island, NY), were eachadded at a concentration of 2 �g/mL. After 20 min of incubationat 2–8 ◦C while protected from light, the wells were washed with100 �L DPBS and the plate was centrifuged. A volume of 220 �L of0.05% (v/v) formaldehyde in DPBS solution was added to each wellto fix the cells after aspiration of the supernatant. Fraction of cellsexpressing gH (reported as relative infectivity yield, %) was mea-sured using the Guava Express Plus protocol on the Guava EasyCyteflow cytometer.

3. Results and discussion

3.1. In situ sonication to disrupt mammalian cells in 96-wellplates: method development

Three sonication instruments, including the Covaris E210, theSonicMan, and the Hielscher UIP250MTP were tested for mechani-cal disruption of uninfected MRC-5 cells cultivated in 96-well platesin situ. In these preliminary studies, the SonicMan and HielscherUIP250MTP did not provide consistent cell lysis in wells across the

plate, with several wells containing intact cells or cell sheet aggre-gates. The Covaris E210 was able to disrupt cells effectively basedon microscopic observation (data not shown); therefore we focusedfurther development on this instrument.

M. Ly et al. / Journal of Biotechnology 184 (2014) 146–153 149

Table 1Effect of Covaris AFA treatment on MRC-5 cell lysis. Microscopic observations of uninfected MRC-5 cells in 96-well plate wells treated with different intensity and duty cycleat a constant treatment of 20s are captured.

Intensity % Duty cycle Treatment time (s) Observed power (W) Lysis description Visual appearanceb

0 0 20 0 No lysis–intact cells and cell sheets Fig. 2A0.1 0.1 20 <0.5 Cell sheet with large patches of lysis Fig. 2C0.1 0.5 20 <0.5 Cell sheet with small patches of lysis Fig. 2B1 1 20 <0.5 Cell lysis with cell aggregates Fig. 2D2 0.5 20 <0.5 Complete cell lysis Fig. 2E5 0.1 20 <0.5 Complete cell lysis Fig. 2E0.5 5 20 1 Cell lysis with cell aggregates Fig. 2D1 2 20 1 Cell lysis with cell aggregates Fig. 2D1 5 20 2 Complete cell lysis Fig. 2E2 2 20 2 Complete cell lysis Fig. 2E1 10 20 4 Complete cell lysis Fig. 2E2 10 20 8 Complete cell lysis Fig. 2E8 5 20 14 Complete cell lysisa Fig. 2F

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Adaptive focused acoustics (AFA) on the Covaris was then fur-her evaluated to carefully determine a suitable combination ofntensity settings and duty cycle that would enable complete lysisdetachment of monolayer with no intact cells visible) of uninfected

ig. 2. Light microscopy images of uninfected MRC-5 cells cultured in 96-well plates andysis. (A) Untreated, no lysis, (B) cell sheet with large patches of lysis, (C) cell sheet with

omplete cell lysis with damage to plate. Arrows in panels B and C, highlight patches of ly

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MRC-5 cells in 96-well plates. In order to complete processing ofa 96-well plate in approximately 30 min, treatment time for eachwell was limited to a maximum of 20 s. For this fixed 20 s treat-ment, the intensity settings and duty cycle were varied to result

treated under different Covaris AFA conditions that result in different degrees ofsmall patches of lysis, (D) cell lysis with cell aggregates, (E) complete cell lysis, (F)sis and arrow in panel F indicates damage to the plate.

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50 M. Ly et al. / Journal of Bio

n observed power ranging from less than 0.5–14 W (Table 1).he state of the MRC-5 cell monolayer was observed using lighticroscopy and the conditions tested resulted in variable degrees

f lysis, ranging from intact cell sheets at the low end of the powerpectrum to complete cell lysis at the high end of the power spec-rum (Fig. 2). At the highest levels of intensity and duty cycleettings (i.e. at observed power of 14 W) undesirable damage tohe plastic plates was even observed, thus restricting the range ofvailable power. However, lower observed power (<0.5 W) was suf-cient for detachment of the monolayer and complete lysis of cells.mong the parameters varied and ranges tested, cell lysis was mostensitive to changes in the intensity levels and showed very littleesponse to variation in the duty cycle (Table 1).

Several operational issues were encountered during AFA cellysis in 96-well plates and had to be addressed prior to furtherevelopment (data not shown). To prevent well-to-well contam-

nation and minimize loss of sample from splashing that occursuring AFA treatment, a sealing mat was applied to the 96-welllate prior to AFA treatment and a quick centrifugation was imple-ented after treatment. Air pockets formed between the 96-well

late and Covaris water bath contact and were observed to gener-te inconsistent cell lysis results. To address this, a small hole wasrilled into the top of each 96-well plate prior to AFA treatment tonsure that no air pocket was created when the 96-well plate wasubmerged in the water bath. Some bystander cell lysis was alsobserved in untreated wells that were adjacent to wells treated atigh intensity or for long duration with observed power exceeding

W. Under the gentle lysis conditions chosen (<0.5 W), this effectas minimal and was further alleviated by setting the well station-

ry (without rotation) and by ensuring randomization of conditionscross the plate with replicates at different locations where possi-le. Using an intensifier and adjusting the Z-offset so that the focalone (4 mm diameter) was fully contained within a well (6.35 mmiameter) further minimized this phenomenon.

.2. Adaptive focused acoustics to recover cell-associated VZVnfectivity

The Covaris settings resulting in complete lysis of uninfectedRC-5 cells (see Table 1 for settings) were then applied to VZV-

nfected MRC-5 cells and microscopically confirmed to result inimilar lysis profiles (data not shown). Recovery of infectious virusrom VZV-infected MRC-5 cells was then evaluated across differ-nt Covaris AFA settings spanning a range of powers (<0.5–14 W;able 2) that provided complete cell lysis (Table 1). As Table 2ndicates, the greatest relative infectivity yield was at the low-st power settings tested. Increased power beyond these settings>0.5 W) resulted in reduced relative infectivity yield presumablyue to detrimental effects on the labile virus. Hence, an inten-ity of 5, duty cycle of 0.1%, and treatment time of 20 s providedufficient power for complete cell disruption with high recoveryf infectivity. These AFA settings were chosen to be applied forll further studies and will be referred to as the “optimal AFAethod”.To confirm the degree of cell disruption with VZV-infected MRC-

cells, the particle size distributions of treated samples wereollected using a CASY® particle counter (Supplementary Fig. 1A).

reference sample using a traditional tip-horn to sonicate bead-arvested MRC-5 cells cultured and infected with VZV in rollerottles was used for comparison (Supplementary Fig. 1B). The parti-le size distribution of an “optimal AFA method” treated sample wasimilar to that of a tip-sonicated sample, with a significant major-

ty of particle counts (>80%) in the 3.3–6 �m range (particles lesshan 3.3 �m were not measured). Less than 10% of the total countsere greater than 20 �m, demonstrating that the optimal AFA sett-

ngs are capable of reducing particle size to significantly less than

VZV infectivity yield kinetics for different MOIs (PFU/cell) is plotted versus timepost-infection. Harvested samples were diluted 2-fold and tested using the virusinfectivity yield assay.

18 �m (typical diameter of trypsinized MRC-5 cells). This indicatedthat the optimal AFA method resulted in effective disruption ofVZV-infected MRC-5 cells cultivated in 96-well plates.

MRC-5 cells were then infected at MOI ranging from 1:4 to1:128 PFU/cell to determine if the virus infectivity yield using thechosen AFA conditions matched expected results from variationin virus input. The optimal AFA method was applied to sam-ples harvested between 12 and 52 h post infection (hpi). Theselysed samples were then diluted and tested in the virus infectiv-ity yield assay. Since a relative comparison was desired, data fromseveral dilutions were analyzed to select an appropriate dilutionfactor where the assay response was detectable but not saturatedfor all harvest times post-infection (Supplementary Fig. 2). Basedon this analysis, the relative infectivity yield from 2-fold dilutedsamples is presented in Fig. 3. The observed kinetics of infec-tion demonstrated a strong correlation with MOI, i.e. higher MOIresulted in a faster increase in harvested infectivity for MOI in therange of 1:16–1:128 PFU/cell (Fig. 3). For the highest MOI tested(1:4–1:16 PFU/cell), the kinetics of harvested virus infectivity yieldwere similar, presumably indicating a saturation of virus input. Apeak in harvested infectivity was reached for MOI greater than1:64 PFU/cell by 44 hpi (earlier for even higher MOI), after whichthere is a decrease in harvested infectivity, with the most pro-nounced decrease observed for the highest MOI range (greater than1:8 PFU/cell). Among the conditions tested, only the 1:128 PFU/cellMOI condition did not result in >50% relative virus infectivity yieldby the last harvest time point of 52 hpi. Overall, the data reproducesa typical VZV infectivity profile with initial kinetics responsive tovirus input followed by a peak and a decline dependent on MOI.While the specific MOI range and culture conditions were slightlydifferent, the data presented here follows the general trends pre-viously reported for VZV by Provost et al. (1994, 1997) and Singhviet al. (1996).

To assess well-to-well variability for the 96-well plate celldisruption method, MRC-5 cells were first infected with VZV inrandomly positioned wells (n = 4) across 3 replicate plates. CovarisAFA treated samples were tested in the virus infectivity yield assay.The standard deviation in relative virus infectivity yield acrosswells for the three plates (Supplementary Fig. 3A; 1–10%) and

overall (Supplementary Fig. 3B; 3–15%) give a sense of variability.However, this variability is attributable to the variability from thecell disruption method and variability from the infectivity assay.To deconvolute, virus infectivity yield was measured for tip-horn

M. Ly et al. / Journal of Biotechnology 184 (2014) 146–153 151

Table 2Effect of Covaris AFA treatment on virus infectivity yield. MRC-5 cells infected with VZV MOI of 1:30 PFU/cell were harvested at 24 h post-infection and treated using differentintensity, duty cycle, and treatment time settings. Relative virus infectivity yield (%) data for sample dilution factor of 2 is shown.

Intensity % Duty cycle Treatment time (s) Observed power (W) Lysis description Relative infectivity yield (%)b

5 0.1 20 <0.5 Complete cell lysis 63.32 5 5 4.5 Complete cell lysis 34.08 5 20 14 Complete cell lysisa 13.18 5 180 14 Complete cell lysisa 4.0

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Fig. 4. Effect of MRC-5 plant cell density on VZV infectivity yield in 96-well plates.(A) Time-profile of MRC-5 cell density over 7 days post-plant. Error bars represent±1 standard error of mean of well replicates, n = 6. (B) Time-profile of relative VZVinfectivity yield at different plant cell densities. MRC-5 cells were infected at anMOI of 1:30 PFU/cell 3 days post-plant. Harvested samples were diluted 4-fold andtested using the virus infectivity yield assay. Error bars represent ±1 standard error

a Damage to bottom of plate occurred at this setting.b Values from samples diluted 2-fold.

onicated VZV infected MRC-5 cell samples (cells disrupted inulk outside the Covaris instrument) at different dilutions across

plates (1 well/plate). The standard deviation in the virus infec-ivity yield for the tip-horn sonicated samples (Supplementaryig. 3C; 5–15%) were comparable to the overall standard devia-ion in the Covaris AFA treated samples. This suggests that theell disruption method in 96-well plates contributes less to theariability observed when compared to the virus infectivity yieldssay. Accordingly, multiple replicate measurements were takenn the virus infectivity yield assay in experiments to address assayariability.

.3. Process development case study: application of AFA tovaluate effect of MRC-5 cell density on cell-associated VZVnfectivity yield

To demonstrate the applicability of the in situ cell disruption andntracellular product recovery method in a process developmentetting, we evaluated cell density effects on VZV production. MRC-5ells were planted at a range of cell densities and monitored for cellrowth (Fig. 4A) and cell cycle status (Supplementary Fig. 4A). Theeak cell density of ca. 5.0 × 105 cells/cm2 was attained on differ-nt days post-plant for all plant cell densities ≥0.4 × 105 cells/cm2.ell density did not reach the peak target by day 6 post-plant forhe lower plant cell densities of 0.1–0.2 × 105 cells/cm2. Cell cyclenalysis of cells harvested at different days post-plant revealedhat at higher plant cell densities (≥0.8 × 105 cells/cm2), the frac-ion of cells in the S-phase was generally lower 1 day post-plantnd decreased further over time. At lower plant cell densities≤0.4 × 105 cells/cm2), the fraction of cells in S-phase increased forhe first few days and then began to decrease. The widest variationn cell cycle states (fraction of cells in the S-phase ranging from 10o 40%) was observed on day 3 post-plant, so this time was choseno infect cells with VZV to understand cell density and cell cycleffects on virus production.

MRC-5 cells planted at different densities were infected withZV at MOI of 1:30 PFU/cell 3 days post-plant. The resulting cellensities at infection were not linearly correlated with the plantell densities (Supplementary Fig. 4B), possibly due to media-elated limitations at higher cell densities. Virus infectivity yieldrom cells planted at a cell density of 0.2 × 105 cells/cm2 yieldedhe highest virus infectivity yield across the range of time pointsFig. 4B). Higher cell densities (>0.2 × 105 cells/cm2) or lower cellensities (0.1 × 105 cells/cm2) yielded lower virus infectivity. Inrder to compare virus productivity on a per infected cell basis,elative virus infectivity yield was normalized by the infectionell density (Fig. 4C). Following this analysis, the highest perell virus yield was observed at the lowest infection cell densitycorresponding to 0.1 × 105 cells/cm2 plant density). This analy-is provided some indication that the environment or cell stateesulting from infection at lower cell densities could enable produc-

ion of more infectious virus. Taken together, the virus infectivityield seems to depend on both the number of cells at time ofnfection and the cell specific productivity affected by “cell state”.hese observations are similar to results seen with adenovirus and

of mean of assay well replicates, n = 3. (C) Relationship between MRC-5 infection celldensity, cell cycle status (fraction of cells in S-phase) and relative VZV infectivityyield (absolute and normalized to infection cell density) at peak infectivity timingpost-infection (38 hpi). Error bars represent ±1 standard error of mean of assay wellreplicates, n = 3.

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aculovirus propagation in cell culture (Bernal et al., 2009; Shent al., 2010) and are likely representative of many virus cultureystems.

The evaluation of cell density effects on virus infectivityield was a typical process development exercise. However, thisork was accomplished much more efficiently using a 96-wellicroscale HT method in place of the roller bottle based man-

facturing process. For instance, the number of conditions andeplicates tested in Fig. 4 (5 infection conditions, 5 harvest times,nd 3 replicates for each condition) would have taken 4 scientistsnd multiple experiments (∼155 roller bottles per experiment),ut was accomplished in a single experiment with a single per-on here. The use of an HT virus infectivity yield assay (Gatest al., 2009; Joelsson et al., 2010) also enabled this work, whereashe typical plaque assay could not have supported the nec-ssary analytical throughput. Reagent utilization, including cellulture media, was also significantly reduced using 96-well plates200 �L per condition) instead of roller bottles (>100 mL per con-ition). A newer version of the Covaris E210 instrument fromhe same manufacturer (Covaris LE220) claims capability to pro-ess 8 wells simultaneously and should reduce overall processingime, making this method even more attractive for HTS applica-ions.

. Conclusions

A microscale cell disruption method was desired for analysisf detergent sensitive intracellular or cell-associated products in aigh-throughput (HT) setting. We have identified adaptive focusedcoustic (AFA) disruption as a viable technology for this applica-ion. Low energy mechanical disruption of Varicella-Zoster virusnfected MRC-5 cells using AFA was demonstrated to be effectiven lysis and release of infectious cell-associated virus in a 96-welllate format in situ. Using this lysis procedure it was verified that

nfectivity from released virus was dependent on the input virusn a dose-dependent manner (MOI effect), and infection kinetics

ere demonstrated to be similar to previously reported work. Thenfectivity from released virus increases with time post-infectiono reach a peak value and then shows a decline in infectivity, asould be expected for in vitro production of a labile virus. This

ysis procedure was used to demonstrate that dividing MRC-5 cellsay be more productive (on a per cell basis) than non-dividing

ells. However, the overall virus infectivity yield was a functionf both the cell cycle status and the number of cells at the timef infection. This AFA method provides a HT approach to disruptammalian cells, especially when detergents cannot be used for

roduct recovery.

cknowledgements

We would like to thank James Laugharn at Covaris, Inc. and Marcenger at Merck & Co., Inc. for providing input into the develop-ent of the AFA process used for adherent cell culture in 96-well

lates. We would also like to thank Jennifer McMackin and Danoelsson for providing the procedure and support for the semi-utomated flow cytometric virus infectivity assay. We appreciatend thank Warren Pilbrough, James Warren, and James Wagner forheir review and feedback.

ppendix A. Supplementary data

Supplementary material related to this article can be found,n the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014.4.030.

logy 184 (2014) 146–153

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