The authors would like to thank biolitec Research GmbH (Jena, Germany) for kindly providing us with the liposomal mTHPC formulation, with special thanks to Susanna Gr&#228;fe and Arno Wiehe for their help and technical expertise. We also would like to thank Kerstin Reidy, who generated a large part of the results and was coresponsible for its publication11.
This work was supported by grants from the Schweizerischer Verein Balgrist, the University of Zurich, the Krebsliga Zurich as well as by a grant from the Walter L. and Johanna Wolf Foundation, Zurich, Switzerland and a grant from EuroNanoMed ERA-NET &#8260;SNF Swiss National Science Foundation 31NM30-131004 &#8260; 1. This work was also supported by the HSM (Highly Specialized Medicine) program for Musculoskeletal Oncology of the Kanton Zurich.

1.&#160;Comparison of the Uptake of mTHPC in the Respective Low and Highly Metastatic HOS and 143B OS Cell Lines
<ol>
	<li>Prepare cell culture medium containing DMEM, Ham F12, and heat inactivated fetal calf serum in a ratio 4.5:4.5:1. 
	Note: The liposomal formulation of the PS mTHPC was dissolved in water at a final concentration of 1.5 mg/ml mTHPC.</li>
	<li>Plate 0.2 x 106 HOS and 143B cells/well in 6-well plates (triplicates for each condition) and let the cells adhere overnight. 
	Note: the number has to be adjusted to individual cell lines. A confluency of 80-90% is most suitable at the time of the experiment.</li>
	<li>On the following day, incubate the cells with a fixed concentration of mTHPC for different time periods (time dependent uptake), or with increasing concentrations of mTHPC during a fixed time period (dose dependent uptake).</li>
	<li>Incubate the cells with mTHPC for specific time points or concentrations of mTHPC. 
	Note: In this study, both the HOS and 143B cell lines were incubated with 0.6 &#181;g/ml mTHPC for 0, 2.5, 5, or 10 hr, or with 0, 0.6, 2.5, and 10 &#181;g/ml mTHPC for 5 hr. The applied time periods and dosages may vary depending on the cell line that is used.</li>
	<li>Remember to always work in the dark or in dimmed light conditions&#160;to prevent direct light exposure of the cells.</li>
	<li>After incubation of the cells at the indicated conditions, aspirate the medium and wash the cells three times with PBS. Next, detach the cells with 0.5 ml trypsin/EDTA and count the cells.</li>
	<li>After detachment, centrifuge the cells at 400 x g for 5 min, aspirate the medium and resuspend the cells in PBS to a final density of 200,000 cells/ml.</li>
	<li>Pipette 100 &#181;l of the obtained cell suspension (i.e.&#160;20,000 cells) in a 96-well plate and measure the fluorescence of these cells in a fluorescence spectrophotometer . The settings for mTHPC fluorescence measurements are: 417 nm for the excitation and 652 nm for the emission.</li>
	<li>In order to calculate the amount of internalized mTHPC per cell, normalize the relative fluorescence unit (RFU) for the cell number and cell volume as follows:</li>
	<li>Prepare a standard curve using different concentrations of mTHPC in PBS from 0-4 &#181;g/ml (use triplicate measurements). Pipette 100 &#181;l of each dilution in a 96-well plate and measure the fluorescence of the wells using the settings described under step 1.6.</li>
	<li>Divide the RFU (obtained for 20,000 cells) by the slope of the linear standard curve of mTHPC, which gives the concentration of mTHPC (in &#181;g/ml) in the well.</li>
	<li>Divide this mTHPC concentration by 10 to get the total amount of mTHPC in one well (in &#181;g, representing 100 &#181;l of cell suspension) and divide this value by the volume of 20,000 cells.</li>
	<li>Calculate the volume with the formula for a sphere: (4/3 x &#960;) x r3. Obtain the radius r by measuring the diameter of 20 trypsinized cells under a microscope, and dividing this value by 2.</li>
	<li>Finally, normalize all the values to those obtained for HOS cells after 10 hr of incubation (dose dependent uptake) or treated with 10 &#181;g/ml mTHPC (time dependent uptake), which were set to 100%.</li>
</ol>
2.&#160;Measurement of Phototoxicity of PS In Vitro
<ol>
	<li>Seed 3,000 cells/well in a 96-well plate (triplicates for each concentration of mTHPC used) and let them adhere overnight. Similarly prepare an additional 96-well plate and treat the cells as described below, but then put them aside for phase contrast imaging. 
	Note: the number has to be adjusted for each individual cell line. A confluency of 50-60% at the time of the experiment is ideal.
	<ol>
		<li>Keep in mind to add the corresponding dark toxicity control of cells for each of the mTHPC concentrations. These are cells that will be incubated in the absence or presence of selected concentrations of mTHPC, but will not be illuminated.</li>
	</ol>
	</li>
	<li>On the following day, incubate the cells with different concentrations of mTHPC (0, 0.0001, 0.001, 0.01, 0.03, 0.075, 0.15, 0.3, 0.6, 1.25, 2.5, 5,&#160;and 10 &#181;g/ml) depending on the cell lines and keep them in the dark for 5 hr.
	<ol>
		<li>Refer to Section 1.3.2 
		Note: The incubation time before illumination depends on the cell line and on the PS used, therefore it is advisable to test different concentrations and different time points in separate experiments.</li>
	</ol>
	</li>
	<li>Following incubation for the indicated time period, wash the cells twice with PBS and add new fresh medium without mTHPC.</li>
	<li>Illuminate the cells with a 652 nm diode laser, specific for the mTHPC absorption spectrum (see Figure 1).
	<ol>
		<li>Set the laser to 21.88 mW/cm2 power at a height of 13.5 cm distance from the cells, in order to get an energy dose of 5 J/cm2. Illuminate the cells for 230 sec. Do not illuminate the dark toxicity control cells (with and without mTHPC). 
		Note: Always wear glasses to protect the eyes from the laser light used for excitation of the PS.</li>
	</ol>
	</li>
	<li>After illumination, keep the cells in the dark at 37 &#186;C for 24 hr. Subsequently, add water soluble tetrazolium (WST-1) reagent (10 &#181;l/100 &#181;l of medium).</li>
	<li>Three hours after the addition of the WST-1 reagent, measure the absorbance of individual wells of the 96-well plate at 415 nm.</li>
	<li>In order to calculate the percentage of surviving cells, set the mean of the values obtained for the non mTHPC treated cells for each condition to 100% (i.e.&#160;illuminated, without mTHPC and nonilluminated, without mTHPC).</li>
</ol>
3.&#160;Estimation of Cell Number by Cell Counting
<ol>
	<li>Seed 20,000 cells/well in a 24-well plate and let them adhere overnight.</li>
	<li>Incubate the cells for 5 hr with mTHPC (0.001, 0.003, 0.15, 0.6, and 1.25 &#181;g/ml) and illuminate them as described above.</li>
	<li>Collect the medium, wash the cells once with PBS, trypsinize them and collect them by centrifugation at 400 x g for 5 min.</li>
	<li>After centrifugation, aspirate the medium and resuspend the cells in 200 &#181;l of fresh medium.</li>
	<li>Count the cells in a Neubauer chamber.</li>
</ol>

<ol>
	<li>Juarranz, A., Jaen, P., Sanz-Rodriguez, F., Cuevas, J., Gonzalez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+cancer.+Basic+principles+and+applications.">Photodynamic therapy of cancer. Basic principles and applications.</a> Clin. Transl. Oncol. 10, 148-154 (2008).</li><li>Agostinis, P., 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=Photodynamic+therapy+of+cancer:+an+update.">Photodynamic therapy of cancer: an update.</a> CA Cancer. 61, 250-281 (2011).</li><li>D'Adamo, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appraising+the+current+role+of+chemotherapy+for+the+treatment+of+sarcoma.">Appraising the current role of chemotherapy for the treatment of sarcoma.</a> Semin. Oncol. 38 Suppl 3, 19-29 (2011).</li><li>Allison, D. C., 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+meta-analysis+of+osteosarcoma+outcomes+in+the+modern+medical+era.">A meta-analysis of osteosarcoma outcomes in the modern medical era.</a> Sarcoma. , (2012).</li><li>Anninga, J. 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=Chemotherapeutic+adjuvant+treatment+for+osteosarcoma:+where+do+we+stand.">Chemotherapeutic adjuvant treatment for osteosarcoma: where do we stand.</a> Eur. J. Cancer. 47, 2431-2445 (2011).</li><li>Bacci, G., 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=Pattern+of+relapse+in+patients+with+osteosarcoma+of+the+extremities+treated+with+neoadjuvant+chemotherapy.">Pattern of relapse in patients with osteosarcoma of the extremities treated with neoadjuvant chemotherapy.</a> Eur. J. Cancer. 37, 32-38 (2001).</li><li>Briccoli, 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=Resection+of+recurrent+pulmonary+metastases+in+patients+with+osteosarcoma.">Resection of recurrent pulmonary metastases in patients with osteosarcoma.</a> Cancer. 104, 1721-1725 (2005).</li><li>Milla Sanabria, 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=Direct+and+indirect+photodynamic+therapy+effects+on+the+cellular+and+molecular+components+of+the+tumor+microenvironment.">Direct and indirect photodynamic therapy effects on the cellular and molecular components of the tumor microenvironment.</a> Biochim. Biophys. Acta. 1835, 36-45 (2013).</li><li>Mitra, S., Foster, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photophysical+parameters,+photosensitizer+retention+and+tissue+optical+properties+completely+account+for+the+higher+photodynamic+efficacy+of+meso-tetra-hydroxyphenyl-chlorin+vs+Photofrin.">Photophysical parameters, photosensitizer retention and tissue optical properties completely account for the higher photodynamic efficacy of meso-tetra-hydroxyphenyl-chlorin vs Photofrin.</a> Photochem. Photobiol. 81, 849-859 (2005).</li><li>Ma, L., Moan, J., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+new+photosensitizer,+meso-tetra-hydroxyphenyl-chlorin,+for+use+in+photodynamic+therapy:+a+comparison+of+its+photobiological+properties+with+those+of+two+other+photosensitizers.">Evaluation of a new photosensitizer, meso-tetra-hydroxyphenyl-chlorin, for use in photodynamic therapy: a comparison of its photobiological properties with those of two other photosensitizers.</a> Int. J. Cancer. 57, 883-888 (1994).</li><li>Reidy, K., Campanile, C., Muff, R., Born, W., Fuchs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTHPC-mediated+photodynamic+therapy+is+effective+in+the+metastatic+human+143B+osteosarcoma+cells.">mTHPC-mediated photodynamic therapy is effective in the metastatic human 143B osteosarcoma cells.</a> Photochem. Photobiol. 88, 721-727 (2012).</li><li>Triesscheijn, M., Ruevekamp, M., Aalders, M., Baas, P., Stewart, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+sensitivity+of+microvascular+endothelial+cells,+fibroblasts+and+tumor+cells+after+in+vitro+photodynamic+therapy+with+meso-tetra-hydroxyphenyl-chlorin.">Comparative sensitivity of microvascular endothelial cells, fibroblasts and tumor cells after in vitro photodynamic therapy with meso-tetra-hydroxyphenyl-chlorin.</a> Photochem. Photobiol. 80, 236-241 (2004).</li><li>Triesscheijn, M., Baas, P., Schellens, J. H., Stewart, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+oncology.">Photodynamic therapy in oncology.</a> Oncologist. 11, 1034-1044 (2006).</li><li>Burch, S., 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=Treatment+of+canine+osseous+tumors+with+photodynamic+therapy:+a+pilot+study.">Treatment of canine osseous tumors with photodynamic therapy: a pilot study.</a> Clin. Orthop. Relat. Res. 467, 1028-1034 (2009).</li></ol>

The authors declare&#160;no competing financial interests or conflicts of interest.

To achieve optimal cytotoxicity in response to PDT, it is crucial to choose the right laser light settings and incubation times. The here described procedures are consistent and efficient to determine PS uptake and to quantify PDT induced cytotoxicity in vitro. Using the specific absorption wavelengths of the PS mTHPC, the cellular PS uptake can be determined in a direct manner, and the PS can be activated to generate cytotoxic reactive oxygen species.
Using this in vitro set...

Today&#8217;s state of the art treatment of osteosarcoma (OS), a primary bone tumor, encompasses a combination of neo adjuvant chemotherapy and surgery. This treatment regimen revealed an increase in the survival rate of patients with localized disease from approximately 20% before the use of chemotherapy, to currently between 60-70%3,4. However, in the last two decades, the overall survival of OS patients with local disease has plateaued4,5. Moreover, 30-40% of these patients relapse within 3 years after diagnosis and patients with metastatic disease continue to have a poor survival of 20-30%4,6,7. To improve the outcome of these patients, new therapeutic strategies need to be developed.
Photodynamic Therapy (PDT), a rather novel anticancer therapy, uses light of a specific wavelength for excitation of a photosensitizer (PS), which accumulates in the tumor cells after its injection into the bloodstream. Laser light excitation of the PS generates oxygen radicals in the presence of oxygen, which induce cytotoxic reaction in tumor cells and cell death. Besides this primary mechanism, two additional PDT evoked biological processes contribute to reduced tumor growth: PDT causes vasoconstriction and thrombus formation of the tumor microvasculature and, consequently local hypoxia and anoxia inside the tumor, leading to tumor infarction. Finally, PDT injured and dying tumor cells trigger a local immune response, a rather unique feature of PDT. This involves the complement system and the activation of antigen presenting dendritic cells8. Thus, conditions are created for the presentation of tumor antigens with subsequent activation of lymphoid cells, leading to tumor specific immunity.
So far, PDT has been used to treat several types of soft tissue tumors and hyperplasia&#8217;s, such as actinic keratosis, Barrett&#8217;s esophagus, endobronchial tumors, bladder cancer, basal cell carcinomas, and palliative treatment of head and neck cancer2. The treatment is known to induce local, large scale necrosis with only little side effects, and thus has the potential to selectively eradicate tumor tissue. Despite these advantages, the application of PDT remains technically more demanding than the administration of chemotherapeutic drugs. In order to achieve maximal efficacy, the PS concentration, light exposure time and total light energy transfer need to be optimized. This can be done in in vivo experiments, but, because of the relative large number of parameters that need to be optimized, it is more efficient to initially determine optimal conditions in vitro.
In the experiments described below, we tested the in vitro efficacy of PDT using the PS 5,10,15,20-tetrakis(meta-hydroxyphenyl)chlorin, abbreviated mTHPC (Figure 1A). mTHPC is the active substance in the medicinal product Foscan, which is currently used in the clinic for palliative treatment of head and neck cancer. It is one of the most potent PS, inducing massive cell damage already at low concentrations, and it was demonstrated to be superior to other PS in terms of tissue penetration9,10. Its light absorption spectrum (Figure 1B) shows two prominent peaks, one at 417 nm and a second at 652 nm, which are used for tissue localization of accumulating PS and for PDT induction, respectively.
Currently, a liposomal formulation for mTHPC is under development. Here, we describe the procedures to quantify the uptake of this liposomal formulation, and to perform PDT in two human OS cell lines; the low metastatic HOS and the high metastatic 143B cells. Some of the data presented here have been reported earlier11. The approach described here enables us to study the effect of a metastatic phenotype on PDT efficacy. 143B cells, orthotopically injected into the hind limbs of immune deficient SCID mice cause intratibial metastasizing primary tumors, a model closely mimicking the human metastasizing disease. Thus, the proposed in vitro experiments are perfectly suitable to assess the optimal PDT settings to be later used in in vivo experiments.

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:251px;">PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-883</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HAM F12</td>
			<td>PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">heat-inactivated fetal calf serum</td>
			<td>GIBCO, Basel, Switzerland</td>
			<td style="width:127px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:291px;">mTHPC</td>
			<td style="width:251px;">biolitec research GmbH, Jena, Germany</td>
			<td style="width:127px;"></td>
			<td style="width:287px;">As this liposomal formulation is originating from R &#38; D 
			no catalogue number is available at the moment. Stock: 1.5mg/ml; provided in a 9:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG; &#62;99% purity)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectramax Gemini XS plate reader</td>
			<td style="width:251px;">Molecular Devices, Sunnyvale, CA</td>
			<td style="width:127px;">ID# 861</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Microscope Zeiss Observer.Z1</td>
			<td style="width:251px;">Axio Observer, Axio Vision Release 4.6.3 SP1, Jena Germany</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ceralas PDT 652 nm Laser</td>
			<td style="width:251px;">Biolitec AG, Jena, Germany</td>
			<td style="width:127px;">LD652nm2W400u</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Water-soluble tetrazolium (WST) reagent</td>
			<td style="width:251px;">Roche Diagnostics AG, Rotkreuz, Switzerland</td>
			<td style="width:127px;">1644807</td>
			<td></td>
		</tr>
	</tbody>
</table>

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

With the here reported techniques, we investigated mTHPC-based PDT in human OS cells. First, the time&#160; and dose dependent uptake of mTHPC was investigated in the low metastatic HOS and in the highly metastatic 143B OS cell lines. mTHPC uptake can be assessed by measuring the fluorescence of mTHPC with a fluorescence spectrophotometer (Figure 2, reproduced with permission from Reidy et al.11). Figure 2A illustrates the uptake of mTHPC in a time dependent manner. T...

Watch this Scientific Journal Video about Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro at JoVE.com

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

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12.8K Views.  Balgrist University Hospital, Zurich, Switzerland.  Il protocollo qui riportato permette di valutare l'efficacia della terapia fotodinamica (PDT) in linee cellulari e l'ottimizzazione delle impostazioni PDT prima di applicare la terapia in modelli animali. 

Video: Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.