This research was funded by grant number PID2019-105128RB-I00 to RSA, JMB, and AA, and PGC2018-095795-B-I00 to PFS and RML, both funded by MCIN/AEI/10.13039/501100011033 and grant numbers B31_20R (RSA, JMA, and AA) and E35_17R (PFS and RML) and funded by Gobierno de Arag&#243;n. The work of RSA was supported by a grant from the Asociaci&#243;n Espa&#241;ola Contra el C&#225;ncer (AECC) PRDAR21487SOLE. The authors would like to acknowledge the use of Servicio General de Apoyo a la Investigaci&#243;n-SAI, Universidad de Zaragoza.

NOTE: All culture media and buffer compositions are specified in Table 1. Prior to cybrid generation, both mitochondrial and nuclear DNA profiles from the donor and recipient cells must be typed to confirm the presence of genetic differences in both genomes between cell lines. In this study, a commercially available L929 cell line and its derived cell line, L929dt, which was spontaneously generated in our laboratory (see13 for more information) were used. These cell lines present two differences in the sequence of their mt-Nd2 gene which can be used to confirm the purity of mtDNA once the cybridization process has been finished13. In this case, the purity of the nuclear background was confirmed by antibiotic sensitivity, since, contrary to L929dt cells, L929 were resistant to geneticin.
1. Mitochondrial depletion by rhodamine 6G treatment (recipient cells)
NOTE: The first step for successful cybrid generation is to completely and irreversibly abolish mitochondrial functions in the recipient cells. For this purpose, it is necessary to previously determine, for each cell line, the appropriate concentration and treatment duration with rhodamine 6G. This adequate concentration should be just below drug-induced cell death (the highest that does not kill the cells during the treatment). Perform the following once the optimal conditions are defined.
<ol>
	<li>Seed 106 cells in a 6-well plate using complete culture medium (see Table 1 for details) and treat them daily with the optimal concentration of rhodamine 6G for 3-10&#160;days, depending on the cell line. Traditional concentrations range from 2 to 5 &#181;g/mL for 3-10 days20,21. In this study, for L929-derived cells, 2.5 &#181;g/mL rhodamine 6G for 7 days was the selected treatment13,22.</li>
	<li>To keep the cells alive, supplement the cell culture medium with 50 &#181;g/mL of uridine and 100 &#181;g/mL of pyruvate and renew it every 24 h.</li>
	<li>After treatment and prior to fusion, change the medium of rhodamine 6G-treated cells to complete cell culture medium without rhodamine 6G, and leave them in this medium for 3-4 h in an incubator at 37 &#176;C with 5% CO2. 
	NOTE: The rhodamine 6G toxic effect is irreversible, and cells with non-functional mitochondria should not recover20. Thus, after treatment, cells that do not receive functional mitochondria should die even in uridine-supplemented culture medium. Therefore, no nuclear selection should be necessary. However, a control fusion of rhodamine 6G-treated cells without mitochondria is recommended.</li>
</ol>
2. Expansion and mitochondrial isolation (donor cells)
<ol>
	<li>Expand the mitochondria donor cells during the time lapse of rhodamine 6G treatment to obtain around 25 x 106 cells.</li>
	<li>On day 7, harvest exponentially growing cells in a 50 mL tube and collect them by centrifugation at 520 x g for 5 min at room temperature (RT). Wash the cells 3x with cold phosphate-buffered saline (PBS) and sediment them by centrifugation at 520 x g for 5 min at RT. From now on, perform all the steps of mitochondrial extraction at 4 &#176;C, using cold reagents and keeping the tubes with cells or mitochondria on ice.</li>
	<li>After the third centrifugation, discard the supernatant by aspiration using a glass pipette coupled to a vacuum pump and resuspend the packed cells in a volume of hypotonic buffer equal to 7x the cell pellet volume. Then, transfer the cell suspension into a homogenizer tube and let the cells swell by incubating them on ice for 2 min.</li>
	<li>Break the cell membranes by performing 8 to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rpm. 
	NOTE: The step of cell membrane disruption can vary between cell types; thus, it has to be optimized for each cell type.</li>
	<li>Add the same volume of hypertonic buffer to the cell suspension (7x the cell pellet volume) to generate an isotonic environment.</li>
	<li>Transfer the homogenate into a 15 mL tube and centrifuge it in a fixed rotor at 1,000 x g for 5 min at 4 &#176;C. Then, collect only 3/4 of the supernatant leaving a large margin from the pellet, to avoid contamination with nuclei or intact cells, and transfer it to another tube. Repeat the same process twice. Note that the supernatant must be kept and the pellet discarded.</li>
	<li>Save the mitochondrial fraction (supernatant). Transfer it into 1.5 mL tubes and centrifuge at maximum speed (18,000 x g) for 2 min at 4 &#176;C.</li>
	<li>Discard the supernatant and wash the mitochondria-enriched pellet with buffer A, combining the content of the two tubes into one and centrifuging at the same conditions as described in step 2.7. Repeat the same process until all the material is in only one tube.</li>
	<li>Make an additional wash with 300 &#181;L of buffer A and quantify the mitochondrial protein concentration using the Bradford assay23. For each cybridization assay, the optimal amount of mitochondria for the transfer procedure has to be determined (in our case, a concentration between 10-40 &#181;g of mitochondrial protein per 106 cells).</li>
	<li>Simultaneously, prepare the rhodamine 6G-pretreated cells for the fusion by collecting them in a 15 mL tube and centrifuging at 520 x g for 5 min at RT. Note that the pellet acquires a neon pink color due to rhodamine 6G treatment.</li>
	<li>To ensure that both mitochondrial function abolishment in receptor cells as well as organelle purification from donors have been properly performed, seed a small number of rhodamine 6G-treated cells and isolated mitochondria using the complete culture medium in a 6-well plate and culture them for a month to check that no surviving cells remain in any of the wells (Figure 2).</li>
	<li>In parallel, evaluate the absence of nuclei contaminants in the mitochondrial fraction by immunodetection of nuclear proteins (i.e., lamin beta, histone H3, etc.) or by quantitative polymerase chain reaction (qPCR) amplification of a nuclear gene (i.e., SDH, 18S rRNA, etc.). 
	NOTE: To avoid contaminations, it is recommended to perform all the steps in aseptic conditions working under a laminar flow hood.</li>
</ol>
3. Fusion and cybrid generation
<ol>
	<li>To proceed with the fusion, carefully add 106&#160;of the rhodamine 6G-treated cells to the isolated mitochondria pellet (10-40 &#181;g of mitochondrial protein) and centrifuge at 520 x g for 5 min to allow the cells to mix with the mitochondria.</li>
	<li>Add 100 &#181;L of polyethylene glycol (PEG, 50%) and gently resuspend the pellet for 30 s. Then, allow to rest untouched for another 30 s.</li>
	<li>Finally, transfer the mix into a 6-well plate with fresh complete cell culture medium and place in the incubator at 37 &#176;C with 5% CO2. After a few days (usually 1 week), transmitochondrial cybrids should start growing (Figure 2), giving rise to clones that can be individually selected or mixed in a pool prior to their analysis.</li>
</ol>
4. Verification of both mitochondrial and nuclear background
NOTE: Once the new cell line has been established and cells begin to grow exponentially, the purity of their mitochondrial and nuclear DNAs must be verified. Thus, the original cell lines should harbor different mutations or polymorphisms within their genomes to make them recognizable.
<ol>
	<li>Total DNA isolation
	<ol>
		<li>Isolate the genomic DNA of all cell lines used for cybrid generation by employing a commercial genomic DNA extraction kit (see Table of Materials) or by performing a standard protocol using phenol-chloroform-isoamyl alcohol extraction and alcohol precipitation24.</li>
	</ol>
	</li>
	<li>mtDNA evaluation (Figure 3) 
	NOTE: Several techniques, such as sequencing, restriction fragment length polymorphism (RFLP) analysis, or allele-specific qPCR, can be performed to analyze the purity of mtDNA. To confirm the presence of mtDNA sequence variations by RFLP, follow the next protocol steps.
	<ol>
		<li>Amplify an mtDNA fragment containing the nucleotide change by PCR.
		<ol>
			<li>Here, L929dt cells present an mtDNA mutation at position 4206, within an mt-Nd2 gene (m.4206C&#62;T) that is absent in L929 cells. To confirm the presence of this substitution in the transmitochondrial cybrids, amplify a 397 bp fragment by PCR using a standard protocol and the following primers: 1) 5&#39;-AAGCTATCGGG 
			CCCATACCCCG-3&#39;&#160;(positions 3862-3884) and 2) 5&#39;-TAATCAGAAGTGGAATGGGGCG -3&#39; (positions 4236-4258).</li>
		</ol>
		</li>
		<li>Analyze the presence of the desired nucleotide substitution by RFLP, using a specific endonuclease that recognizes the sequence change and generates a different cut pattern for both cell lines. 
		NOTE: When the L929dt mtDNA variant is present (4206T), the amplicon obtained in step 4.2.1 contains two restriction sites for SspI (see Table of Materials) that produces three DNA fragments of 306 bp, 52 bp, and 39 bp. The restriction site that generates the 52 and 39 bands is disrupted when the wild-type (WT) version C4206 is present, and a new band of 91 bp appears. Therefore, an internal control for full digestion for SspI is included in the analysis. The digestion reaction is performed at 37 &#176;C, following the manufacturer&#39;s instructions.</li>
		<li>Separate the restriction fragments by electrophoresis and compare the band pattern.
		<ol>
			<li>Once the digestion is performed, analyze the obtained restriction fragments by electrophoresis in 10% polyacrylamide-Tris-borate-EDTA (TBE) gels (see Table 1 for 1x TBE composition). Run the electrophoresis at 80 V for 1 h at RT. Visualize the DNA fragments after gel staining in a solution of ethidium bromide in 1x TBE for 15 min at RT (see Table 1 for gel staining solution composition).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Nuclear DNA genotyping 
	NOTE: Nuclear DNA genotyping must be performed using the previous DNA extraction sample employed for RFLP analysis.
	<ol>
		<li>Amplify 16 loci (D21S11, CSF1PO, vWA, D8S1179, TH01, D18S51, D5S818, D16S539, D3S1358, D2S1338, TPOX, FGA, D7S820, D13S317, D19S433, and AMEL) by using a pool of commercial-specific oligonucleotides (see Table of Materials).</li>
		<li>Perform electrophoresis by using a genetic analyzer in order to separate the fragments previously obtained. 
		NOTE: Once amplified, the different loci are analyzed by electrophoresis using a capilar system (see Table of Materials). The fragments are separated in a 50 cm long capilar filled with a commercial polymer. Cathode and anode buffers are also commercially available, as shown in the Table of Materials. Electrophoresis is performed at 19.5 kV for 20 min at 60 &#176;C.</li>
		<li>Use bioinformatical tools to determine the alleles corresponding to each amplified locus (see Table of Materials).</li>
		<li>Compare the data obtained in step 4.3.3 with the nuclear DNA profile database (Table 1), to check if the nuclear DNA profile matches with the mitochondria receptor cell line profile (Figure 4).</li>
	</ol>
	</li>
</ol>

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

The authors declare no conflicts of interest.

Since Otto Warburg reported that cancer cells shift their metabolism and potentiate &#34;aerobic glycolysis&#34;3,4 while reducing mitochondrial respiration, the interest in the role of mitochondria in cancer transformation and progression has grown exponentially. In recent years, mutations in the mtDNA and mitochondrial dysfunction have been postulated as hallmarks of many cancer types25. To date, numerous studies have analyzed the mtDNA ...

Reprogramming energy metabolism is a hallmark of cancer1 that was observed for the first time by Otto Warburg in the 1930s2. Under aerobic conditions, normal cells convert glucose into pyruvate, that then generates acetyl-coA, fuelling the mitochondrial machinery and promoting cellular respiration. Nevertheless, Warburg demonstrated that, even under normoxic conditions, most cancer cells convert pyruvate obtained from the glycolysis process into lactate, shifting their way to obtain energy. This metabolic adjustment is known as the &#34;Warburg effect&#34; and enables some cancer cells to supply their energetic demands for rapid growth and division, despite generating ATP less efficiently than the aerobic process3,4,5. In recent decades, numerous works have supported the implication of metabolism reprogramming in cancer progression. Hence, tumor energetics is considered an interesting target against cancer1. As a central hub in energetic metabolism and in the supply of essential precursors, mitochondria play a key role in these cell adaptations that, to date, we only partially understand.
In line with the above, mitochondrial DNA (mtDNA) mutations have been proposed as one of the possible causes of this metabolic reprogramming, which could lead to an impaired electron transport chain (ETC) performance6 and would explain why some cancer cells enhance their glycolytic metabolism to survive. Indeed, it has been reported that mtDNA accumulates mutations within cancer cells, being present in at least 50% of tumors7. For example, a recent study carried out by Yuan et al. reported the presence of hypermutated and truncated mtDNA molecules in kidney, colorectal, and thyroid cancers8.&#160;Moreover, many works have demonstrated that certain mtDNA mutations are associated with a more aggressive tumor phenotype and with an increase in the metastatic potential of cancer cells9,10,11,12,13,14,15,16.
Despite the apparent relevance of the mitochondrial genome in cancer progression, the study of these mutations and their contribution to the disease have been challenging due to limitations in the experimental models and technologies currently available17. Thus, new techniques to understand the real impact of mitochondria DNA in cancer disease development and progression are needed. In this work, we introduce a protocol for transmitochondrial cybrid generation from suspension-growing cancer cells, based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria, that overcomes the main challenges of traditional cybridization methods18,19. This methodology allows the use of any nuclei donor regardless of the availability of their corresponding &#961;0 cell line and the transfer of mitochondria from cells that, following the traditional techniques, would be difficult to enucleate (i.e., non-adherent cell lines).

<table><tbody><tr><td>3500XL Genetic Analyzer&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>4406016</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>08-772-1B</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>AmpFlSTR Identifiler Plus PCR Amplification Kit</td><td>ThermoFisher Scientific</td><td>4427368</td><td></td></tr><tr><td>Anode Buffer Container 3500 Series</td><td>Applied Biosystems</td><td>4393927</td><td></td></tr><tr><td>Boric acid</td><td>PanReac</td><td>131015</td><td></td></tr><tr><td>Bradford assay</td><td>Biorad</td><td>5000002</td><td></td></tr><tr><td>Cathode Buffer Container 3500 Series</td><td>Applied Biosystems</td><td>4408256</td><td></td></tr><tr><td>Cell culture flasks</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>DMEM high glucose</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>EDTA</td><td>PanReac</td><td>131026</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Sigma-Aldrich</td><td>E8751</td><td></td></tr><tr><td>Geneticin</td><td>Gibco</td><td>10131027</td><td></td></tr><tr><td>Homogenizer Teflon pestle</td><td>Deltalab</td><td>196102</td><td></td></tr><tr><td>L929 cell line</td><td>ATCC</td><td>CCL-1</td><td></td></tr><tr><td>MiniProtean Tetra4 Gel System</td><td>BioRad</td><td>1658004</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>PCR primers</td><td>Sigma-Aldrich</td><td>Custom products</td><td></td></tr><tr><td>Polyacrylamide Solution 30%</td><td>PanReac</td><td>A3626</td><td></td></tr><tr><td>Polyethylene glycol</td><td>Sigma-Aldrich</td><td>P7181</td><td></td></tr><tr><td>POP-7</td><td>Applied Biosystems</td><td>4393714</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td></td></tr><tr><td>QIAmp DNA Mini Kit</td><td>Qiagen</td><td>51306</td><td></td></tr><tr><td>Rhodamine-6G</td><td>Sigma-Aldrich</td><td>R4127</td><td></td></tr><tr><td>Serum Fetal Bovine</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>SspI</td><td>New England Biolabs</td><td>R3132</td><td></td></tr><tr><td>Streptomycin/penicillin</td><td>PAN biotech</td><td>P06-07100</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S3089</td><td></td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Tris</td><td>PanReac</td><td>P14030b</td><td></td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3750</td><td></td></tr></tbody></table>

transmitochondrial cybrid generation using cancer cell lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

After following the above-presented protocol, a homoplasmic cybrid cell line with a conserved nuclear background but with a new mitochondria genotype should be obtained, as represented in the schematics in Figure 1 and Figure 2. The purity of the mitochondrial and nuclear DNA present in the cybrids can be confirmed by RFLP, as shown in Figure 3, and by nuclear DNA genotyping analysis, as shown in Figure 4</stron...

Watch this Scientific Journal Video about Transmitochondrial Cybrid Generation Using Cancer Cell Lines at JoVE.com

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

transmitochondrial-cybrid-generation-using-cancer-cell-lines

Research

JoVE Journal

Cancer Research

2.3K Views.  University of Zaragoza. Dit onderzoek heeft tot doel de bijdrage van mitochondrion aan kankerprogressie en metastase beter te begrijpen. De transmitochondriale cybride generatie kan ons helpen om op te helderen hoe en op welk niveau mitochondriale genotypes betrokken zijn bij de agressiviteit van een tumorcel. Een van de technologieën om informatie te verkrijgen over de mitochondriale rol bij kanker is cybride generatie.We combineren deze tool met een verscheidenheid aan andere methoden, waaronder donatieve ...

Video: Auteur in de schijnwerpers: Transmitochondriale cybrid-generatie met behulp van kankercellijnen  

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Genetics

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes.
The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called &#34;transmitochondrial cybrids.&#34; Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho0 cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human ...

Biolistic Transformation of Yeast Mitochondria for Mutagenetic Analysis

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

Author Spotlight: Advancing Techniques and Discoveries in Protein Synthesis and Assembly Through Innovative Mitochondrial Research

This work explains how to transform yeast mitochondria using a biolistic method. We also show how to select and purify the transformants and how to introduce the desired mutation in the target position within the mitochondrial genome.

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Biology

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

Investigating Mitochondrial Transfer in MSC and ARPE19 Co-Cultures

Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods.
We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.

Author Spotlight: Bidirectional Mitochondrial Transfer between MSCs and Retinal Pigment Epithelium Cells &#8212; Pathways and In Vivo Challenges

Here, we present a protocol including mitochondrial tracing, direct co-culture procedures of mesenchymal stem cells (MSCs) and retinal pigment epithelial cells (ARPE19), as well as the methods for observing and statistically analyzing tunneling nanotubes (TNT) formation and mitochondrial transfer to characterize mitochondrial exchange via TNTs between MSCs and ARPE19 cells.

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important ...

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

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