Direct Visualization Techniques Research Articles

Abstract P1-07-02: New high-quality HER2 IQFISH pharmDx™ assay with a ½ working day procedure and high concordance to HER2 FISH pharmDx™

Abstract Introduction: HER2 assessment for selection of patients that may benefit from HER2 targeting treatment can be performed by either immunohistochemistry (IHC), fluorescence or chromogen in situ hybridization (FISH or CISH). FISH is a robust and reliable technique for direct visualization and quantitative determination of gene amplifications, deletions and translocations in human cancer cells, but FISH protocols are time-consuming and involve toxic reagents. By introducing a new non-toxic ethylene carbonate based hybridization buffer that perform with very short hybridization times, the total FISH assay time on breast cancer tissue sections can be reduced from the traditional 16–20 hours to 3½–4½ hours. Material and methods: The new Dako HER2 IQFISH pharmDx™ was compared with Dako HER2 FISH pharmDx™ in a comparative study on 120 breast cancer specimens, and reproducibility of the HER2 IQFISH pharmDx™ assay was investigated in a study comprising 3 different sites and a total of 6 different observers. Samples for the comparative study was evaluated by Dako HercepTest™ to include all IHC scoring groups (0, 1+, 2+, 3+). Slides were stained according to manufacturer's instructions using microwave oven for heat pretreatment and RTU pepsin for 3–5 minutes at 37 °C. Hybridization was performed for 2 hours when using HER2 IQFISH pharmDx™ and for 17–20 hours when using HER2 FISH pharmDx™ Kit. All slides were blinded before evaluation. HER2 status was classified as “Non-amplified” when the HER2/CEN17 ratio < 2.0 and “Amplified” when the HER2/CEN17 ratio ≥ 2.0. Results: The new non-toxic hybridization buffer introduces a major safety improvement since formamide is no longer needed. Significantly shorter hybridization times are required to generate the same signal intensity (1–2 hour hybridization versus overnight). HER2 IQFISH pharmDx™ was compared with the traditional HER2 FISH pharmDx™ in a comparative study on 120 breast tissue specimens of human breast carcinoma. The preliminary data on HER2 status for 78 patients obtained by the two assays gave an overall agreement of 98.7% with lower and upper 95% confidence limits at 94.2% and 99.9%. The Kappa value was 0.96 (95% CL: 0.89–1.00). The p-value for McNemars test was 1.00 indicating absence of bias between the two assays. Disagreement between the two assays was observed for one specimen - a heterogeneous tissue with a small amplified area. Data from the reproducibility study that included site-to-site variation, day-to-day variation and inter-observer variation showed that the assay has a high degree of reproducibility. Conclusion: The validation studies of the new HER2 IQFISH pharmDx™ showed a very high concordance to the traditional HER2 FISH pharmDx™ and also that the assay is robust and reproducible. Reduction of the overall assay time from a two-day to a half-day procedure for HER2 FISH, offers more flexible laboratory routines and same day reporting for all working days of the week, which could be used for fast and simultaneous FISH and IHC answers and improved patient care. Taken together, the study demonstrates the potential of a new revolutionary platform that enables optimization and acceleration of FISH analysis to the benefit of cancer patients and laboratory personnel. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-02.

Photodynamic Therapy in Cholangiocarcinoma

Most patients with hilar cholangiocarcinoma have unresectable disease and require palliation with biliary stenting to alleviate symptoms. Although chemotherapy and radiotherapy have been ineffective, many studies have suggested that the use of photodynamic therapy (PDT) may improve quality of life and perhaps survival in these patients. The ability of PDT to transmit through bile makes it a unique platform for intervention in the biliary tree. This article addresses the clinical implications of endoscopic biliary drainage, explores past and present study findings with PDT for cholangiocarcinoma, and offers an introduction to the novel techniques for direct visualization.

Sampling and detection of skin Propionibacterium acnes: Current status

A connection between acne vulgaris and Propionibacterium acnes has long been suggested. Over the years, several human skin microbiota sampling methods have been evolved and applied, e.g. swab, scrape, extraction techniques including cyanoacrylate gel sampling as well as punch biopsy. Collected samples have been processed following various methodologies ranging from culture studies to probe labelling and molecular analysis. Direct visualization techniques have recently shown the existence of anatomically distinct skin P. acnes populations: epidermal and follicular. P. acnes biofilms appear to be a common phenomenon. Current sampling approaches target different skin populations of P. acnes and the presence of microbial biofilms can influence the retrieval of P. acnes. The anatomical considerations must be taken into account while interpreting microbiological data.

P3.05 A New Non-Toxic Hybridization Buffer Reduces the Fish Assay Time From 16-20 Hours to 3½ Hours Without Compromising the Quality

ABSTRACT Fluorescence in-situ hybridization (FISH) is a powerful technique for direct visualization and quantitative determination of gene amplifications, deletions and translocations in human cancer cells. It is recognized as a reliable and reproducible method, and FISH assays are often referred to as 'Gold Standard' for determining the patient status with predictive tests for HER2 and ALK. But FISH is also known to have characteristics which limit a more widespread use, including the long assay time (staining and scoring), requirement for a fluorescence microscope and complexity involved in handling small volumes and toxic reagents. Dako has addressed the toxicity and long hybridization time by introducing a new hybridization buffer. The new non-toxic hybridization buffer (IQFISH buffer), which allows for significant reduction in hybridization time, brings the total assay time for a FISH assay down from 16-20 hours to just 3½ hours without compromising the quality. The new Dako HER2 IQFISH pharmDx™ (assay time 3½ hours) has been compared with the traditional Dako HER2 FISH pharmDx™ Kit (assay time 16-20 hours) in a comparative study on 78 breast tissue specimens of human breast carcinoma ( Fig 1 ). Cross tabulation of HER2 status obtained by the two assays gave an overall agreement of 98.7% (95% CI: 94.2%-99.9%). Disagreement between the two assays was observed for one specimen that most likely was a heterogeneous tissue with a very small area of amplification. A similar comparative study on 79 gastric adenocarcinoma specimens showed an overall agreement of 98.7% (95% CI: 94.2%-99.9%). The disconcordant sample was a heterogeneous tissue with a very small area of amplification. A third concordance study on 80 breast cancer tissues compared TOP2A IQFISH pharmDx™ with the traditional TOP2A FISH pharmDx™ Kit ( Fig 2 ). A 100% overall agreement (95% CI: 96.9%-100%) was found. Conclusion: We have developed new non-toxic hybridization buffer that by reduction of the hybridization time from 16 to 2 hours allows for a ½ day FISH protocol. The IQFISH buffer has been validated for HER2 on breast and gastric cancer and for TOP2A on breast cancer. Both assays showed a very high agreement with the traditional 2 day FISH procedure. Download : Download full-size image Fig. 1 . Agreement between the new Dako HER2 IQFISH pharmDx™ and the traditional Dako HER2 FISH pharmDx™ Kit. Download : Download full-size image Fig. 2 . Agreement between the new Dako TOP2A IQFISH pharmDx™ and the traditional Dako TOP2A FISH pharmDx™ Kit.

A diagnostic genetic test for the physical mapping of germline rearrangements in the susceptibility breast cancer genes BRCA1 and BRCA2

The BRCA1 and BRCA2 genes are involved in breast and ovarian cancer susceptibility. About 2 to 4% of breast cancer patients with positive family history, negative for point mutations, can be expected to carry large rearrangements in one of these two genes. We developed a novel diagnostic genetic test for the physical mapping of large rearrangements, based on molecular combing (MC), a FISH-based technique for direct visualization of single DNA molecules at high resolution. We designed specific Genomic Morse Codes (GMCs), covering the exons, the noncoding regions, and large genomic portions flanking both genes. We validated our approach by testing 10 index cases with positive family history of breast cancer and 50 negative controls. Large rearrangements, corresponding to deletions and duplications with sizes ranging from 3 to 40 kb, were detected and characterized on both genes, including four novel mutations. The nature of all the identified mutations was confirmed by high-resolution array comparative genomic hybridization (aCGH) and breakpoints characterized by sequencing. The developed GMCs allowed to localize several tandem repeat duplications on both genes. We propose the developed genetic test as a valuable tool to screen large rearrangements in BRCA1 and BRCA2 to be combined in clinical settings with an assay capable of detecting small mutations.

Nerve Mapping for Prostatectomies: Novel Technologies Under Development

Prostatic neuroanatomy is difficult to visualize intraoperatively and can be extremely variable. Damage to these nerves during prostatectomies may lead to postoperative complications such as erectile dysfunction and incontinence. This review aims to discuss the prostatic neuroanatomy, sites of potential nerve damage during a prostatectomy, and nerve-mapping technologies being developed to prevent neural injury. These technologies include stimulation, dyes, and direct visualization. Nerve stimulation works by testing an area and observing a physiologic response but is limited by the long half-life for an erectile response; examples include CaverMap, ProPep, and optical nerve stimulation. Few nerve dyes have been approved by the Food and Drug Administration (FDA) because of the extensive testing required; examples of nerve dyes include compounds from Avelas and General Electric, fluorescent cholera toxin subunit B, indocyanine green, fluorescent inactivated herpes simplex 2, and Fluoro-Gold. Direct visualization techniques have a simpler FDA approval process; examples include optical coherence tomography, multiphoton microscopy, ultrasound, coherent anti-Stokes Raman scattering. Many researchers are developing several novel technologies that can be categorized as stimulation based, dye-based, or direct visualization. As of yet, none has shown clear evidence to improve surgical outcomes and consequently lack wide adoption. Further development of these technologies may lead to improved complication rates after prostatectomies. Clinically, some technologies have demonstrated utility in predicting the development of complications. By using that information, more aggressive rehabilitation programs may lead to improved long-term function. These technologies can also be applied for research to improve our knowledge of the neuroanatomy and physiology of erection and incontinence.

P2-13-10: Detection, Visualization and High Resolution Physical Mapping of Large Rearrangements by Molecular Combing in the Hereditary Breast Cancer Genes BRCA1 and BRCA2.

Abstract The BRCA1 and BRCA2 genes are involved, with high penetrance, in breast and ovarian cancer susceptibility. About 2% to 4% of breast cancer patients with a positive family history who are negative for BRCA1 and BRCA2 point mutations can be expected to carry large genomic alterations (deletion or duplication) in one of the two genes, and especially BRCA1. However, large rearrangements are missed by direct sequencing. Molecular Combing is a powerful FISH-based technique for direct visualization of single DNA molecules, allowing the entire genome to be examined at high resolution in a single analysis. We have developed a novel genetic test based on Molecular Combing. For that purpose, we designed specific BRCA1 and BRCA2 “Genomic Morse Codes” (GMC), also covering the non-coding regions and including large genomic portions flanking both genes. We developed a measurement strategy for the GMC signals, and validated our approach by blindly testing 10 breast cancer patients with a positive family history and 10 control patients. Large rearrangements, corresponding to deletions and duplications of one or several exons and with sizes ranging from 3 kb to 40 kb, were detected on both genes, including the characterization of 4 new mutations (for BRCA1: Del ex 3, Del ex 24 and Dup ex 3; for BRCA2: Dup ex 17–20). The identified mutations confirmed the results obtained with high-resolution zoom-in aCGH (11 k) in the same patients, with a resolution in the 1–2 kb range. Importantly, the developed GMC allowed to unambiguously localize several tandem repeat duplications on both genes, and to precisely map large rearrangements in the problematic Alu-rich 5'-region of BRCA1. We propose the developed Molecular Combing genetic test as a valuable tool for the screening of tandem repeat duplications, CNVs, and other complex rearrangements in BRCA1 and BRCA2, such as translocations and inversions, to be combined in clinical settings with an essay that allows the detection of point mutations. We see the main application of the developed molecular diagnostic tool as a predictive genetic test. However, we envisage to extended the application of the developed tool as a companion diagnostic test, for instance in the screening of BRCA-mutated cells in the context of the development of PARP inhibitors. Thus, the genetic test may be applied not only to clinical blood samples, but also to circulating cells and heterogeneous cell populations, such as tumor tissues. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-13-10.

Comparison of esophageal placement of Bravo capsule system under direct endoscopic guidance with conventional placement method

Conventional placement of a wireless esophageal pH monitoring device in the esophagus requires initial endoscopy to determine the distance to the gastroesophageal junction. Blind placement of the capsule by the Bravo delivery system is followed by repeat endoscopy to confirm placement. Alternatively, the capsule can be placed under direct vision during endoscopy. Currently there is no published data comparing the efficiency of one method over the other. The objective of this study was to compare the method of Bravo wireless pH device placement under direct visualization with the conventional method. This retrospective study involved 58 patients (29 patients with indirect and 29 patients with direct visualization) who underwent Bravo capsule placement. The physician's endoscopy procedure notes, nurse's notes, post-procedure notes, recovery notes, and pH monitoring results were reviewed. The safety of the procedure, length of the procedure and patient tolerability were evaluated. None of the 58 patients had early detachment of the device or any immediate procedure-related complications. Overall incidence of complications in both groups was similar. No failures due to the technique were noted in either group. The average amount of time taken for the procedure was similar in the two groups. The technique of placing Bravo pH device under direct visualization is as safe and effective as the conventional method. In addition, the direct visualization technique has an added advantage of avoiding a second endoscopic intubation. The length of the procedure is similar between the two techniques.

Comparison of esophageal placement of Bravo capsule system under direct endoscopic guidance with conventional placement method

Background:Conventional placement of a wireless esophageal pH monitoring device in the esophagus requires initial endoscopy to determine the distance to the gastroesophageal junction. Blind placement of the capsule by the Bravo delivery system is followed by repeat endoscopy to confirm placement. Alternatively, the capsule can be placed under direct vision during endoscopy. Currently there are no published data comparing the efficiency of one method over the other. The objective of this study was to compare the method of Bravo wireless pH device placement under direct visualization with the conventional method.Methods:A retrospective study involving 58 patients (29 patients with indirect and 29 patients with direct visualization) who had Bravo capsule placement. The physician endoscopy procedure notes, nurse’s notes, postprocedure notes, recovery notes, and pH monitoring results were reviewed. The safety of the procedures, length of the procedures, and patient tolerability were evaluated.Results:None of the 58 patients had early detachment of the device and had no immediate procedure-related complications. The overall incidence of complications in both the groups was similar. No failures due to the technique were noted in either group. Average amount of time taken for the procedure was similar in both groups.Conclusion:The technique of placing a Bravo pH device under direct visualization is as safe and effective as the conventional method. In addition, there is an added advantage of avoiding a second endoscopic intubation in the direct visualization technique.

Gum elastic bougie-assisted intubation following hemorrhage in the floor of the mouth

Upper airway obstruction caused by postoperative hemorrhages or surgical manipulation is one of the life-threatening complications for which oral and maxillofacial surgeons may have to initiate emergency management of the airway. Therefore, oral and maxillofacial surgeons need to be skilled in emergency airway management. However, intubation under these circumstances may be very difficult or impossible using direct visual laryngoscopy techniques. This case report describes a case of successful intubation assisted by a gum elastic bougie for an asphyxiating patient suffering from a severely compromised upper airway.

Solubility of Flurbiprofen in CO2 and CO2 + Methanol

The solubility of (R,S)-2-(2-fluorobiphenyl-4-yl)propanoic acid (flurbiprofen) in supercritical carbon dioxide and in a mixed solvent consisting of carbon dioxide and methanol at (303.15 and 313.15) K between the pressures of (10.0 and 20.0) MPa is reported. The solubilities were measured using a direct visualization technique. The experimental data is described using the Peng−Robinson equation of state (PR EoS) together with a one-parameter van der Waals mixing rule.

Direct visualization of hetero-enzyme co-encapsulated in mesoporous silicas

In this study, a direct visualization technique was applied to the determination of a hetero-enzyme co-encapsulated in pores of mesoporous silicas in solution. Lipase and trypsin modified with fluorescent dyes, used as model enzymes, were co-encapsulated at the same instant in two kinds of mesoporous silicas – FSM-22 and SBA-15 – with pore diameters of 4.2 and 7.1 nm, respectively. A direct observation of composite materials of a hetero-enzyme and a mesoporous silica by a combination of differential interference contrast and fluorescence microscopy revealed that the enzymes were uniformly dispersed throughout the particles of the mesoporous silicas, because of the successful incorporation of the two enzymes. Moreover, lipase and trypsin co-encapsulated in the mesopores maintained their enzymatic activities in hydrolysis reactions of their substrates (triglyceride and casein, respectively), without loss of their enzymatic activities due to structural degradation.

Permeabilization method forin-situinvestigation of fungal conidia on surfaces

4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in-situ hybridization (FISH) show great potential for the detection of fungal conidia, also conserving the spatial architecture of their colonization. These investigations are often greatly hampered by the complicated wall structure of many fungal taxa. The aim of the present study was to develop an efficient permeabilization strategy for both DAPI staining and the FISH technique, applicable to various fungal species and maintaining their relationships with surfaces. We compared different DAPI staining permeabilization strategies based on alcohol dehydration, surfactants and osmotic shock, tested with Aspergillus niger conidia. Among four permeabilization methods leading to a strong DAPI signal, only one, based on Triton X-100, EDTA and beta-mercaptoethanol followed by hyperosmotic stress, appeared suitable for FISH investigation and was successfully applied to an additional 10 fungal taxa and three environmental samples. The effective permeabilization method, which employed a combination of surfactant and osmotic strategies, was successfully applied as preliminary step in both DAPI staining and the FISH protocol. The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.

Catalase encapsulated in mesoporous silica and its performance

Catalase was confined in a nanospace of about 7 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a catalase-FSM conjugate. That the enzyme was encapsulated in the pores of mesoporous materials was proven by the direct visualization technique. The catalase-FSM showed not only an activity similar to that of the native catalase in the decomposition of H 2O 2 but also much higher stability: specifically, the catalase immobilized in FSM still retained its original activity after being applied 50 times to the decomposition, whereas the native lost its activity after 20 times recycling. Furthermore, the catalase-FSM showed catalase activity even in an organic media in which the native catalase had almost no catalytic activity.

Comparison of Outcomes of Peritoneal Dialysis Catheters Placed by the Fluoroscopically Guided Percutaneous Method versus Directly Visualized Surgical Method

To compare complications in catheters placed by the fluoroscopically guided percutaneous method versus directly visualized surgery. A retrospective cohort analysis was performed. Mechanical complication rate data, including catheter leakage, malfunction, malposition, and bleeding, were compared between the two groups over a 1-year follow-up period. Additionally, exit site infection rates, tunnel infection rates, and peritonitis episodes were evaluated based on the incidence within 30 days of insertion and 30 days to 1 year after insertion. A total of 101 patients were analyzed (52 in the fluoroscopic guidance group, 49 in the direct visualization group). Prevalence of diabetes was similar: 56% in the directly visualized surgery group and 47% in the fluoroscopically guided treatment group (P = .37). Although the difference was not significant, complication rates tended to be higher in the directly visualized surgery group compared with the percutaneous placement group. These included catheter leakage (13% vs 4%; P = .093), malfunction (11% vs 9%; P = .73), malposition (13% vs 6%; P = .20), and bleeding (8% vs 2%; P = .21). There were no differences in early and late exit site infections and tunnel infections. Late peritonitis rates were lower in the percutaneous placement group (20%) than in the direct visualization group (42%) (P = .018). Diabetic patients had approximately six times greater risk of catheter malfunction than nondiabetic patients regardless of method of catheter insertion. Placement of peritoneal dialysis catheters percutaneously with fluoroscopic guidance is as safe as placement with direct visualization techniques.

Electrode Implantation for Deep Brain Stimulation in Dystonia: A Fast Spin-Echo Inversion-Recovery Sequence Technique for Direct Stereotactic Targeting of the Gpi

Deep brain stimulation (DBS) of the globus pallidus internus (GPi) is an effective treatment for medically refractory primary dystonia. We present our technique for direct preoperative visualization of the target using a fast spin-echo inversion-recovery (FSE-IR) sequence. Twenty-three consecutive patients (mean age 41 years, range 9-68 years, male to female ratio 11:12) with severe dystonia were operated using a combination of FSE-IR imaging for direct visualization of the globus pallidus internus with stereotactic, gadolinium-enhanced T1-MPRage images. The complete procedure, including stereotactic MRI, was performed under general anesthesia with propofol and remifentanyl. We used multichannel microdrive systems (Medtronic; Alpha-Omega) to introduce up to five parallel microelectrodes for microelectrode recordings (MER) and test stimulation with the central trajectory directed at the anatomically predefined target. The initial standard coordinates in relation to the mid-commissural point (mid-AC-PC) were as follows: lateral 21 mm, anterior 3 mm, and inferior 2 mm, which were then adapted to the individual case based on direct visualization of the target area and further refined by the intraoperative neurophysiology. In ten patients (43%) atlas-based standard coordinates were modified based on the direct visualization of the GPi in the FSE-IR images (bilaterally in seven patients, unilaterally in three). The modified targets ranged from 18.5 to 23.5 mm (mean 20.76 mm) laterally, 1-7 mm (mean 2.75 mm) anteriorly and 1-2 mm (mean 1.95 mm) inferiorly to the mid-AC-PC. We implanted the permanent electrode based on the results of MER and intraoperative stimulation performed to determine the threshold for pyramidal tract responses on the central trajectory in 67%, medially in 16%, anteriorly in 11%, laterally in 4%, dorsally in 2%. The procedure resulted in excellent clinical benefits (average reduction of the Burke-Fahn-Marsden Dystonia Rating Score (BFMDRS) or the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) were respectively 65.9%, range 20.9-91.4%) within the first year after surgery. Safety was demonstrated by the absence of intracranial bleeding or other surgical complications causing neurological morbidity. Inversion recovery sequences are an excellent tool for direct visualization of the GPi. These images can be fused to stereotactic MRI or CCT and may help to improve anatomical targeting of the GPi for the implantation of DBS electrodes.

Characterization of Human Coronary Sinus Valves by Direct Visualization during Biventricular Pacemaker Implantation

The precise reasons for failure to cannulate the coronary sinus during biventricular device implantation are unknown. Visualization of the coronary sinus ostium during electrophysiology procedures may enhance understanding of how unusual anatomy can affect successful cannulation of the coronary sinus. The aim of this study was to describe the morphology of valves at the coronary sinus ostium (CSO) visualized directly with an illuminated fiberoptic endoscope during implantation of biventricular devices. The coronary sinus anatomy of one hundred consecutive patients undergoing implantation of biventricular devices was investigated using a fiberoptic endocardial visualization catheter (EVC). The CSO was clearly visualized in 98 patients using the EVC. A Thebesian valve was seen in 54% of these. Almost all Thebesian valves were positioned at the inferior (61%) or posterior (33%) aspect of the CSO. Only six patients had Thebesian valves that covered more than 70% of the CSO, but all were successfully implanted with a transvenous left ventricular pacing lead after cannulating the coronary sinus under direct visualization. Over half of patients undergoing biventricular device implantation have identifiable Thebesian valves. Even valves covering the majority of the ostial area may be traversed using direct visualization and modern catheterization techniques.

Interactive visualization of multidimensional coincidence spectra

The paper presents direct visualization techniques of multidimensional nuclear spectra as well as visualization techniques based on projections of embedded subspaces. While the first group of graphical models is limited to four dimensions, the second one can be theoretically extended to any dimension. The presented algorithms of visualization have been implemented in nuclear data acquisition, processing and visualization system developed at the Institute of Physics, Slovak Academy of Sciences. The paper focuses on presentation of nuclear spectra. However the majority of algorithms can be successfully applied for visualization of scalar arrays of other data types.PACS Code: 29.85.+c, 07.05.Rm

Direct imaging of carrier motion in organic transistors by optical second-harmonic generation

Interest in the dynamic behaviour of carriers in organic materials is motivated by possible applications that include organic thin-film transistors, organic electro-luminescent devices and organic photoconductors. It can also provide an insight into the modelling of carrier transport and trapping in organic semiconductors and insulators. Here, we use an advanced second-harmonic generation technique to probe and visualize real carrier motion in organic materials. This is a time-resolved microscopic optical second-harmonic generation technique that allows direct and selective probing of dynamic carrier motion in organic materials. Experiments making use of this technique and using pentacene field-effect transistors have revealed dynamic changes of second-harmonic-generation intensity profiles arising from pentacene. Carrier velocity in organic solids is thus determined from the visualized carrier motion. We anticipate that this direct visualization technique will find wide application in the illustration of space-charge field formation in organic and inorganic materials, including biomaterials and polymers.

A new in vitro wound model based on the co‐culture of human dermal microvascular endothelial cells and human dermal fibroblasts

Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.