This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Note: The following protocol describes the necessary steps to eliminate specific cells using NIR-PIT. Controls and other details about NIR-PIT and cell viability can be found elsewhere18.
1. Conjugation of IR700 to Monoclonal Antibodies (mAb)
<ol>
	<li>Prepare mAb of interest at 2-5 mg/ml in 0.1 M Na2HPO4 (pH 8.6) solution.</li>
	<li>Mix 6.8 nmol of mAb with 30.8 nmol of 10 mM IR700 in 0.1 M Na2HPO4 solution (pH 8.6) in a microcentrifuge tube and incubate at RT for 1 hr, covered with aluminum foil.</li>
	<li>Wash the PD-10 column (see Table of Materials/Equipment) with 15 ml PBS, twice. Load the sample from step 1.2.</li>
	<li>Purify the mixture via the PD-10 column by PBS elution according to manufacturer&#39;s instructions. 
	Note: Here, the elution was along the band color of IR700. For a 300 &#181;l sample, PBS elution fraction is typically between 2.5 ml to 4.4 ml.</li>
	<li>Determine the protein concentration with Coomassie staining by measuring the absorption at 595 nm with a spectrophotometer9. Determine the concentration of IR700 with absorption at 689 nm to confirm the number of fluorophore molecules conjugated to each mAb molecule9. 
	Note: It is important to determine an optimal conjugation number of IR700 molecules in 1 mAb with a spectrophotometer. Generally, around 3 IR700 molecules in 1 mAb molecule is optimal for both in vitro and in vivo work. HPLC and SDS-PAGE methods can be used to confirm whether the mAb and IR700 are bound or not.</li>
	<li>Store at 4 &#176;C after determining the protein concentration.</li>
</ol>
2. Preparation of Mixed 3D Cell Culture (Mixed Spheroid)
<ol>
	<li>Apply sterile water (around 1 ml) into the plate reservoir section of hanging drop plates.</li>
	<li>Prepare various ratios of the cell types of interest &#8212; A431-luc-GFP cells and 3T3-RFP cells &#8212; with each sample containing 5,000 cells total, suspended in 50 &#181;l of culture media. 
	Note: Prepare 1:100, 10: 100, 25:100, 50:100, etc. ratios of cells types of interest in culture media depending on the cell-type.</li>
	<li>Incubate the mix for 5-7 days in the 96 well hanging drop plate in a humidified incubator at 37 &#176;C and 5% carbon dioxide. Change the culture media every 2 days. Note: To make the 3D spheroid precisely, handle the plate gently, as drops containing cells fall off easily before forming 3D shapes.</li>
	<li>Observe the morphology and size of the spheroids using an inverted brightfield microscope at 10X - 40X magnification. Note: Although it depends on cell types and the size of hanging-drop, ensure that the diameter of the spheroid is around 400-600 &#181;m after 7 days of incubation in the 96 well hanging drop plate.</li>
</ol>
3. In Vitro NIR-PIT for Mixed 3D Cell Culture
<ol>
	<li>Change the media of the hanging drop plates to 10 &#181;g/ml antibody-photoabsorber conjugate (APC) containing media, and incubate for 6 hr in a humidified incubator at 37 &#176;C and 5% carbon dioxide.</li>
	<li>After 6 hr incubation, wash the spheroid twice with fresh culture media (phenol red free). Gently transfer the spheroid to a glass-bottomed 50 mm dish with 100 &#181;l fresh phenol red free culture media using a sterile 200 &#181;l pipette tip with the tip cut off. Place one spheroid in each dish.</li>
	<li>Observe the spheroid with an inverted brightfield microscope to detect the change of morphology. To observe the optical reporters (e.g., GFP and RFP) use fluorescence microscope with the following filter settings: GFP&#160;&#8212; 469 nm excitation filter, and 525 nm emission filter; RFP&#160;&#8212; 559 nm excitation filter, and 630 nm emission filter.</li>
	<li>Place the light-emitting diode (LED) above the glass-bottomed dish for irradiation. 
	Note: Spheroids can be exposed to NIR light either on the microscope or in the laminar hood.
	<ol>
		<li>Measure the power density of the NIR-light with an optical power meter9. According to this measurement, irradiate NIR-light via light-emitting diodes (LEDs) which emit light at wavelengths of 670 to 710 nm at 2 J/cm2. 
		Note: LED light can penetrate maximum depth of approximately 5 inches. Cytotoxic effects of NIR-PIT is dependent only on given energy regardless of power density and duration of exposure23.</li>
	</ol>
	</li>
	<li>After irradiation, transfer the spheroid into a new hanging drop plate with 50 &#181;l of fresh culture media and incubate for 1 day in a humidified incubator at 37 &#176;C and 5% carbon dioxide.</li>
	<li>Gently transfer the spheroid to a glass-bottomed dish with 100 &#181;l of fresh culture media (phenol red free) using a sterile 200 &#181;l pipette tip with the tip cut off. Observe the spheroid with a fluorescence microscope 1 day after NIR-PIT using filter settings described in step 3.3.
	<ol>
		<li>Detect dead cells by adding propidium iodide to the media at a final concentration of 2 &#181;g/ml, or by loss of cytoplasmic GFP-fluorescence using a fluorescence microscope (Figure 2B)14,18,22.</li>
	</ol>
	</li>
	<li>Repeat steps 3.1-3.6 if target cells are not completely eliminated.</li>
</ol>

<ol>
	<li>Robinton, D. A., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+induced+pluripotent+stem+cells+in+research+and+therapy.">The promise of induced pluripotent stem cells in research and therapy.</a> Nature. 481 (7381), 295-305 (2012).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+past,+present,+and+future.">Induced pluripotent stem cells: past, present, and future.</a> Cell stem cell. 10 (6), 678-684 (2012).</li><li>Birchall, M. A., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering's+green+shoots+of+disruptive+innovation.">Tissue engineering's green shoots of disruptive innovation.</a> Lancet. 6736 (14), 11-12 (2014).</li><li>Ben-David, U., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumorigenicity+of+human+embryonic+and+induced+pluripotent+stem+cells.">The tumorigenicity of human embryonic and induced pluripotent stem cells.</a> Nat. Rev. Cancer. 11 (4), 268-277 (2011).</li><li>Hanna, J. H., Saha, K., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotency+and+cellular+reprogramming:+facts,+hypotheses,+unresolved+issues.">Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues.</a> Cell. 143 (4), 508-525 (2010).</li><li>Lee, M. -. O., Moon, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+pluripotent+stem+cell-derived+teratoma+formation+by+small+molecules.">Inhibition of pluripotent stem cell-derived teratoma formation by small molecules.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (35), 3281-3290 (2013).</li><li>Miura, K., Okada, Y., 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=Variation+in+the+safety+of+induced+pluripotent+stem+cell+lines.">Variation in the safety of induced pluripotent stem cell lines.</a> Nat. Biotechnol. 27 (8), 743-745 (2009).</li><li>Tang, C., Lee, A. 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=An+antibody+against+SSEA-5+glycan+on+human+pluripotent+stem+cells+enables+removal+of+teratoma-forming+cells.">An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells.</a> Nat. Biotechnol. 29 (9), 829-834 (2011).</li><li>Mitsunaga, M., Ogawa, M., Kosaka, N., Rosenblum, L. T., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+-+selective+in+vivo+near+infrared+photoimmunotherapy+targeting+specific+membrane+molecules.">Cancer cell - selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules.</a> Nat. Med. 17 (12), 1685-1691 (2011).</li><li>Mitsunaga, M., Nakajima, T., Sano, K., Kramer-Marek, G., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+in+vivo+target-specific+cancer+cell+death+after+near+infrared+photoimmunotherapy.">Immediate in vivo target-specific cancer cell death after near infrared photoimmunotherapy.</a> BMC Cancer. 12 (1), 345 (2012).</li><li>Nakajima, T., Sano, K., Mitsunaga, M., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+in+vivo+acute+necrotic+cancer+cell+death+induced+by+near+infrared+photoimmunotherapy+using+fluorescence+lifetime+imaging.">Real-time monitoring of in vivo acute necrotic cancer cell death induced by near infrared photoimmunotherapy using fluorescence lifetime imaging.</a> Cancer Res. 72 (18), 4622-4628 (2012).</li><li>Sano, K., Mitsunaga, M., Nakajima, T., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+cytotoxic+effects+of+photoimmunotherapy+assessed+by+18F-FDG+PET.">Acute cytotoxic effects of photoimmunotherapy assessed by 18F-FDG PET.</a> J. Nucl. Med. 54 (5), 770-775 (2013).</li><li>Sato, K., Watanabe, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoimmunotherapy:+Comparative+effectiveness+of+two+monoclonal+antibodies+targeting+the+epidermal+growth+factor+receptor.">Photoimmunotherapy: Comparative effectiveness of two monoclonal antibodies targeting the epidermal growth factor receptor.</a> Mol. Oncol. 8 (3), 620-632 (2014).</li><li>Sato, K., Nagaya, T., Mitsunaga, M., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+photoimmunotherapy+for+lung+metastases.">Near infrared photoimmunotherapy for lung metastases.</a> Cancer Lett. 365 (1), 112-121 (2015).</li><li>Sato, K., Hanaoka, H., Watanabe, R., Nakajima, T., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+Infrared+Photoimmunotherapy+in+the+Treatment+of+Disseminated+Peritoneal+Ovarian+Cancer.">Near Infrared Photoimmunotherapy in the Treatment of Disseminated Peritoneal Ovarian Cancer.</a> Mol. Cancer Ther. 14 (8), 141-150 (2014).</li><li>Sato, K., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoimmunotherapy+of+Gastric+Cancer+Peritoneal+Carcinomatosis+in+a+Mouse+Model.">Photoimmunotherapy of Gastric Cancer Peritoneal Carcinomatosis in a Mouse Model.</a> PloS one. 9 (11), 113276 (2014).</li><li>Sato, K., Nagaya, T., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+Infrared+Photoimmunotherapy+in+the+Treatment+of+Pleural+Disseminated+NSCLC+Preclinical+Experience.">Near Infrared Photoimmunotherapy in the Treatment of Pleural Disseminated NSCLC Preclinical Experience.</a> Theranostics. 5 (7), 698-709 (2015).</li><li>Sato, K., Nakajima, T., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cell+elimination+in+vitro+and+in+vivo+from+tissues+and+tumors+using+antibodies+conjugated+with+a+near+infrared+phthalocyanine.">Selective cell elimination in vitro and in vivo from tissues and tumors using antibodies conjugated with a near infrared phthalocyanine.</a> RSC Adv. 5, 25105-25114 (2015).</li><li>Watanabe, R., Hanaoka, H., 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=Photoimmunotherapy+Targeting+Prostate-Specific+Membrane+Antigen:+Are+Antibody+Fragments+as+Effective+as+Antibodies.">Photoimmunotherapy Targeting Prostate-Specific Membrane Antigen: Are Antibody Fragments as Effective as Antibodies.</a> J. Nucl. Med. 56 (1), 140-144 (2014).</li><li>Nakajima, T., Sano, K., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+efficacy+of+Photoimmunotherapy+(PIT)+using+a+cocktail+of+antibody+conjugates+in+a+multiple+antigen+tumor+model.">Improving the efficacy of Photoimmunotherapy (PIT) using a cocktail of antibody conjugates in a multiple antigen tumor model.</a> Theranostics. 3 (6), 357-365 (2013).</li><li>Shirasu, N., Yamada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+antitumor+effect+of+CEA-targeted+photoimmunotherapy.">Potent and specific antitumor effect of CEA-targeted photoimmunotherapy.</a> Int J Cancer. 135 (11), 1-14 (2014).</li><li>Sato, K., Nagaya, T., Nakamura, Y., Harada, T., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+photoimmunotherapy+prevents+lung+cancer+metastases+in+a+murine+model.">Near infrared photoimmunotherapy prevents lung cancer metastases in a murine model.</a> Oncotarget. 6 (23), 19747-19758 (2015).</li><li>Nakajima, T., Sato, 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=The+effects+of+conjugate+and+light+dose+on+photo-immunotherapy+induced+cytotoxicity.">The effects of conjugate and light dose on photo-immunotherapy induced cytotoxicity.</a> BMC cancer. 14 (1), 389 (2014).</li><li>Klimanskaya, I., Rosenthal, N., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derive+and+conquer:+sourcing+and+differentiating+stem+cells+for+therapeutic+applications.">Derive and conquer: sourcing and differentiating stem cells for therapeutic applications.</a> Nat. Rev. Drug Discov. 7 (2), 131-142 (2008).</li><li>Burmester, G. R., Feist, E., D&#246;rner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+cell+and+cytokine+targets+in+rheumatoid+arthritis.">Emerging cell and cytokine targets in rheumatoid arthritis.</a> Nat. Rev. Rheumatol. 10 (2), 77-88 (2014).</li><li>Pardoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blockade+of+immune+checkpoints+in+cancer+immunotherapy.">The blockade of immune checkpoints in cancer immunotherapy.</a> Nat. Rev. Cancer. 12 (4), 252-264 (2012).</li></ol>

The authors have nothing to disclose.

We demonstrate a method of specific cell elimination from a mixed 3D cell culture without damage to non-target cells by using NIR-PIT. So far, there is no practical method of cell elimination once the tissue is established or after transplantation. Thus, NIR-PIT is a promising method to accomplish this. This technique could also be utilized in vivo18,22, since APCs show similar pharmacokinetics as mAb itself. The target cell type can be adapted with various APC. Various antibodies or antibody...

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, and there is an urgent need for a cell elimination method in the tissue engineering field. Nowadays in the field of regenerative medicine, tissue cultures using embryonic stem cells (ES), pluripotent stem cells (PSCs), or induced pluripotent stem cell (iPS) are promising materials1-3.
Although this tissue regeneration is promising, contamination with unwanted cells is a major concern. Moreover, there is a safety concern of tumorigenicity after transplantation4,5. Although many studies have focused on these issues to eliminate specific cells, especially in regenerative medicine6-8, no practical method has been developed.
Near infrared photoimmunotherapy (NIR-PIT) is a treatment based on an antibody-photoabsorber conjugate (APC). An APC consists of a cell-specific monoclonal antibody (mAb) and a photoabsorber, IR700. IR700 is a hydrophilic silica-phthalocyanine derivative and does not induce phototoxicity by itself9. IR700 is covalently conjugated to the antibody via amide residues on the side chain of lysine molecules. The APC binds target molecules on the cell membrane and then induces nearly immediate cell necrosis after exposure to NIR light at 690 nm. During the exposure to NIR-light, the cellular membrane ruptures leading to cell death9-14. NIR-PIT has proven to be effective with multiple antibodies or antibody fragments, including anti-EGFR, anti-HER2, anti-PSMA, anti-CD25, anti-mesothelin, anti-GPC3, and anti-CEA15-21. Therefore, NIR-PIT can be used against a wide variety of target molecules. Moreover, NIR-PIT is a well-controlled treatment that allows selective treatment of specific regions by restricting the NIR-light irradiation18,22.
Here, we present a method of specific cell elimination using NIR-PIT from mixed 3D cultures.

<table border="0" cellpadding="0" cellspacing="0" width="1083">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:424px;">IRDye 700DX Ester Infrared Dye</td>
			<td style="width:364px;">LI-COR Bioscience (Lincoln, NE, USA)</td>
			<td style="width:191px;">929-70011</td>
			<td style="width:104px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>SIGMA-ALDRICH (St. Louis, MO, USA)</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G25 column (PD-10)&#160;</td>
			<td>GE Healthcare (Piscataway, NJ, USA)</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coomassie (bradford) Plus protein assay</td>
			<td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td>
			<td>PI-23200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfecta3D 96-Well hanging Drop Plates</td>
			<td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td>
			<td>HDP1096-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical power meter</td>
			<td>Thorlabs (Newton, NJ, USA)</td>
			<td>PM100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED: L690-66-60</td>
			<td>Marubeni America Co. (Santa Clara, CA, USA)</td>
			<td>L690-66-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectibix (panitumumab)</td>
			<td>Amgen (Thousand Oaks, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35mm glass bottom dish, dish size 35mm, well size 10mm</td>
			<td>Cellvis (Mountain View, CA, USA)</td>
			<td>D35-10-0-N</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

To optically monitor the effect of NIR-PIT, the A431 cell line, which overexpresses EGFR, was genetically modified to also express GFP and luciferase (A431-luc-GFP). As a non-target of NIR-PIT, the Balb/3T3 cell line was optically modified to express RFP (3T3-RFP). The APC, panitumumab-IR700 (pan-IR700), was synthesized. Mixed spheroids, which were composed of various ratios of cells (A431-luc-GFP and 3T3-RFP) were fabricated according to this protocol (Figure 1). Repeate...

Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

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7.8K Views.  National Cancer Institute. Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT). 

Video: Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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

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

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

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

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

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Chapters in this video

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

Micropatterning and Assembly of 3D Microvessels

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

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