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

Summary

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).

Abstract

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.

Introduction

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 materials^1-3.

Although this tissue regeneration is promising, contamination with unwanted cells is a major concern. Moreover, there is a safety concern of tumorigenicity after transplantation^4,5. Although many studies ....

Protocol

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)

1. Prepare mAb of interest at 2-5 mg/ml in 0.1 M Na₂HPO₄ (pH 8.6) solution.
2. 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.
3. Wash the PD-10 column (see Table of Materials/Equipment) with 15 ml PBS, twice. Load the sample fr....

Results

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.......

Discussion

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 vivo^18,22, since APCs show similar pharmacokinetics as mAb itself. The target cell type can be adapted with various APC. Various antibodies or antibody.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IRDye 700DX Ester Infrared Dye	LI-COR Bioscience (Lincoln, NE, USA)	929-70011
Na2HPO4	SIGMA-ALDRICH (St. Louis, MO, USA)	S9763
Sephadex G25 column (PD-10)	GE Healthcare (Piscataway, NJ, USA)	17-0851-01
Coomassie (bradford) Plus protein assay	Thermo Fisher Scientific Inc (Waltham, MA, USA)	PI-23200
Perfecta3D 96-Well hanging Drop Plates	3D Biomatrix Inc (Ann Arbor, MI, USA)	HDP1096-8
Optical power meter	Thorlabs (Newton, NJ, USA)	PM100
LED: L690-66-60	Marubeni America Co. (Santa Clara, CA, USA)	L690-66-60
Vectibix (panitumumab)	Amgen (Thousand Oaks, CA, USA)
35 mm glass bottom dish, dish size 35 mm, well size 10 mm	Cellvis (Mountain View, CA, USA)	D35-10-0-N