Proteome Integral Solubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence, and High-Throughput Target Deconvolution

Materials and Reagents

Cell culture

1. Tubes for centrifugation 50 mL and 15 mL (Sarstedt, catalog numbers: 62.559 and 62.554.502)

2. Sterile 25 cm² cell culture flask (Sarstedt, catalog number: 83.3910.002)

3. Cell culture medium [e.g., Dulbecco’s modified Eagle’s medium (DMEM)] (Thermo Fisher Scientific, catalog number: 11685260; original manufacture: Lonza, catalog number: BE12-614F)

4. Fetal Bovine Serum (FBS, Thermo Fisher Scientific, catalog number: 11560636)

5. Penicillin-streptomycin solution (Gibco, catalog number: 15140-122)

6. L-Glutamine (Gibco, catalog number: A2916801)

7. Ca²⁺Mg²⁺-free Dulbecco’s PBS (Cytiva, catalog number: 10462372)

8. TrypLE express enzyme solution (Thermo Fisher Scientific, catalog number: 12605036)

9. Dimethyl sulfoxide solution (DMSO, Merck, catalog number: D8418)

   
   

PISA treatment

1. 0.2 mL PCR tube strips with caps (Thermo Fisher Scientific, catalog number: AB0452)

2. Protein low-binding tubes for centrifugation, 1.5 mL and 2 mL (Thermo Fisher Scientific, catalog numbers: 10708704 and 10718894; original manufacture: Eppendorf, catalog number: 0030108116 and 0030108132)

3. 1.5 mL polypropylene tubes for ultracentrifugation (Beckman Coulter, catalog number: 357448)

4. Ca²⁺Mg²⁺-free Dulbecco’s PBS (Cytiva, catalog number: 10462372)

5. 100× protease inhibitor cocktail solution (Thermo Fisher Scientific, catalog number: 78439)

6. Liquid N₂ in a container

7. Nonidet P-40 (NP40) (Thermo Fisher Scientific, catalog number: 11596671)

8. Milli-Q water

9. Cell lysis buffer (see Recipes)

10. 20% NP40 (see Recipes)

    
    

Soluble protein fraction processing

1. Micro-BCA assay kit (Thermo Fisher Scientific, catalog number: 23235)

2. Dithiothreitol (DTT, Merck, catalog number: D0632)

3. Iodoacetamide (IAA, Merck, catalog number: I6125)

4. Acetone (Thermo Fisher Scientific, catalog number: 10417440)

5. 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS, Merck, catalog number: E9502)

6. Urea (Merck, catalog number: U5378)

7. Lyophilized LysC enzyme (FUJIFILM Wako Pure Chemical Corporation, catalog number: 125-05061)

8. Lyophilized sequencing grade modified trypsin enzyme (Promega, catalog number: V5111)

9. Trypsin Resuspension Buffer (Promega, catalog number: V5111)

10. Tandem Mass Tag (TMT) 10-plex, TMTpro^TM 16-plex, or TMTpro^TM 18-plex isobaric label reagent set (Thermo Fisher Scientific, catalog numbers: 90110, A44520, or A52045)

11. Water-free acetonitrile (Thermo Fisher Scientific, catalog number: 10222052)

12. 50% hydroxylamine solution (Thermo Fisher Scientific, catalog number: 90115)

13. Trifluoroacetic acid (TFA, Merck, catalog number: 302031)

14. Methanol (Skandinaviska Genetec, catalog number: RH1019/2.5)

15. Acetonitrile (ACN, Thermo Fisher Scientific, catalog number: A955-212)

16. Formic acid (FA, Merck, catalog number: 1002641000)

17. pH indicator paper (VWR, catalog number: 85403.600)

18. C18 desalting columns (Waters, catalog number: WAT054960)

19. 0.5 M DTT solution (see Recipes)

20. 0.5 M IAA solution (see Recipes)

21. 20 mM EPPS buffer (pH = 8.2) (see Recipes)

22. 20 mM EPPS buffer (pH = 8.2) including 8M urea (see Recipes)

23. 50% ACN solution (see Recipes)

24. 0.1% TFA (v/v) solution (see Recipes)

25. 2% ACN (v/v) solution including 0.1% TFA (see Recipes)

26. 2% ACN (v/v) solution including 0.1% FA (LC-MS buffer A) (see Recipes)

27. 50% ACN (v/v) solution including 0.1% FA (see Recipes)

28. 80% ACN (v/v) solution including 0.1% FA (see Recipes)

    
    

Peptide separation, mass spectrometry, and proteomics data analysis

1. 28%–30% NH₄OH water solution (Thermo Fisher Scientific, catalog number: 221228-1L-A)

2. Milli-Q water

3. Acetonitrile (Thermo Fisher Scientific, catalog number: A955-212)

4. Reversed-Phase C18 guard column (Waters, catalog number: 186007769)

5. High pH Reversed-Phase C18 Column (Waters, catalog number: 186003621)

6. 0.1% FA in water (Thermo Fisher Scientific, catalog number: 10188164)

7. 0.1% FA in ACN (Thermo Fisher Scientific, catalog number: 10118464)

8. Nano trap-column (Thermo Fisher Scientific, catalog number: 164535)

9. C18 EasySpray nLC peptide separation column (Thermo Fisher Scientific, catalog number: ES803A)

10. 20 mM NH₄OH in H₂O (high pH Buffer A) (see Recipes)

11. 20 mM NH₄OH in ACN (high pH Buffer B) (see Recipes)

12. 98% ACN in H₂O including 0.1% FA (LC-MS buffer B) (see Recipes)

Equipment

Cell culture

1. 37 °C 5% CO₂ incubator (Thermo Fisher Scientific, Forma Steri-cycle i160)

2. Laminar flow cabinet (ninoSAFE, class II)

3. Light microscope (ZEISS, Primo Vert)

4. Cell counter (Bio-Rad, model: TC10)

5. Benchtop centrifuge with swinging-bucket rotor for 15 mL tubes (Eppendorf, model: Centrifuge 5804R)

   
   

PISA treatment

1. Thermal cycler (Applied Biosystems, SimpliAmp)

2. Ultracentrifuge (Beckman Coulter, model: Optima XPN-80)

3. Fixed-Angle Titanium Rotor for centrifugation (Beckman Coulter, model: Type 45 Ti)

4. Thermomixer (Eppendorf, model: ThermoMixer C)

5. Vortex (Scientific Industries, model: Vortex-Genie 2)

6. Milli-Q water purification system (Millipore, model: IQ 7000)

   
   

Soluble protein fraction processing

1. Absorbance plate reader (BioTek, Epoch)

2. Benchtop centrifuge for 1.5 mL tubes (Eppendorf, model: Centrifuge 5430R)

3. Manifold for vacuum extraction for small columns (Waters, Extraction Manifold)

4. Speed-Vacuum concentrator (Eppendorf, concentrator plus)

   
   

Peptide separation, mass spectrometry, and proteomics data analysis

1. Mass spectrometer equipped with an EASY ElectroSpray source (Thermo Fisher Scientific, Orbitrap Q Exactive HF or higher)

   Note: This protocol refers to Orbitrap Q Exactive HF. However, Orbitrap Fusion, Orbitrap Fusion Lumos, Orbitrap Exploris 480, and Orbitrap Eclipse can be also used.

2. Nanoflow UPLC system with fraction collector and UV detector (Thermo Fisher Scientific, Ultimate 3000)

3. Capillary HPLC system for peptide high pH C-18 fractionation with fraction collector and UV detector (Thermo Fisher Scientific, Dionex Ultimate 3000)

Software

1. Absorbance plate reader software (BioTek, Gen5 2.09)

2. Q Exactive HF-Orbitrap MS 2.9 build 2926 (Thermo Fisher Scientific)

3. Thermo Scientific SII for Xcalibur (Thermo Fisher Scientific)

4. MaxQuant software (Max Planck Institute of Biochemistry)

5. Proteome Discoverer 2.5 (Thermo Fisher Scientific)

6. Microsoft Office software (Microsoft)

7. Prism (GraphPad)

Procedure

1. Cell culture and ligand treatment

   1. Thaw the cells to be studied from the N₂ stock vials, and wash them in 10–15 mL of their regular cell culture medium (e.g., DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 2 mM L-Glutamine), and sediment cells down by centrifugation according to cell type-specific recommendations (250 × g for 5 min). Discard the supernatant and resuspend the cells in complete growth medium.

   2. The day before the PISA experiment, split the cells in the number of samples as by experimental design, up to 18–25-cm² cell culture flasks containing 5 mL of complete cell growth medium in each flask, and let the cells attach overnight.

   3. Incubate each biological replicate with active ligands to be studied (e.g., drugs, active molecules of interest)—here named “D”—at a biologically active concentration previously tested (e.g., IC₅₀ or similar) using a stock with 1,000–2,000× concentration, with at least three biological replicates per D ligand. Control replicates are to be treated with the volume of the vehicle solution (e.g., DMSO). In the chosen example of PISA multiplexing scheme, five D ligands (D1–D5) with three biological replicates each and three replicates of the vehicle-treated controls are used, for a total of 18 biological samples.

      Note: The number of biological replicates can be higher, as recommended for lower numbers of D ligands to be tested, depending on the number of molecules or conditions to be tested, and up to 18, by using the TMTpro^TM 18-plex label reagent set, which can host up to five ligand-treated or vehicle-treated samples in triplicate.

   4. Incubate treated cells for 30–90 min in a 5% CO₂ cell culture incubator at 37 °C.

   5. At the end of the treatment with each D or vehicle, collect cells from each flask using TrypLE, and wash the cell pellets twice with 10 mL of PBS, followed by centrifugation (250 × g for 5 min).

      
      

2. Protein extraction for PISA assay samples

   1. Resuspend the cell pellet in 750 μL of PBS buffer including protease inhibitors.

   2. Gently mix, resuspend cells homogeneously, and distribute 45 μL in each 0.2-mL tube, for a total of 16 temperature points to be used for each sample.

   3. Perform step B2 for each sample.

   4. Heat each sample at a range of 44 to 59 °C, with 1 °C intervals, and perform the temperature treatment for 3 min at each temperature.

      Notes:

      1. By using a gradient thermal cycler, samples can be treated at different temperatures at the same time.

      2. The temperature range can be varied and tuned according to specific expectations or needs, with virtually unlimited number of temperature points.

   5. Leave the samples at 23 °C for 6 min.

   6. At the end of the 23 °C incubation, snap freeze the samples in liquid N₂.

   7. Thaw all samples at 37 °C, vortex for 10 s, and snap freeze in liquid N₂.

   8. Repeat step B7 four times, for a total of five freeze-thaw cycles.

   9. For each sample and replicate, combine the contents of 16 tubes corresponding to all temperatures of that replicate into one protein low-binding tube, and add 20% NP40 solution, up to a final concentration of 0.4%, which at this point should not resolubilize insoluble proteins.

   10. Incubate all samples shaking at 350 rpm/min at 4 °C, in a thermomixer or a refrigerated room, for 30 min.

   11. Perform ultracentrifugation at 150,000 × g at 4 °C for 30 min, and recover the supernatant of each replicate without disrupting the pellet.

       
       

3. Soluble protein fraction processing

   
   Day 1

   1. Measure the total protein concentration of all samples, using a micro-BCA kit and an absorbance plate reader.

   2. Take the volume corresponding to 50 μg for each sample. To equalize volumes, dilute all samples with PBS up to the volume of the sample with the least concentration. Perform reduction by adding 0.5 M DTT solution to a final concentration of 8 mM, and incubate samples at 55 °C for 45 min.

   3. Add 0.5 M IAA solution to a final concentration of 25 mM, and incubate samples in darkness at 25 °C for 30 min.

   4. Precipitate proteins using cold acetone at -20 °C overnight, at a sample:acetone ratio of 1:6 (v/v).

      
      
   Day 2

   5. Collect the precipitated proteins by centrifugation (10,000 × g, 10 min), remove the supernatant, and air-dry the pellet for 5 min.

   6. Solubilize each pellet in 15 μL of EPPS buffer at pH 8.2 including 8 M urea for 10 min.

   7. Add 14 μL of EPPS buffer to each sample. Dissolve 20 μg LysC powder using 30 μL of EPPS buffer, then add 1 μL of this LysC enzyme solution (equivalent to 0.67 μg of LysC) to each sample, and allow the digestion to proceed gently mixing at 350 rpm/min at 30 °C for 6 h.

   8. Add 90 μL of EPPS buffer to each sample. Dissolve 20 μg lyophilized sequencing grade modified trypsin using 20 μL of trypsin resuspension buffer, then add 1 μL of this trypsin solution (equivalent to 1 μg of trypsin) to each sample, and let the digestion occur gently mixing at 350 rpm/min at 37 °C overnight.

   9. Stop the reaction the day after by placing the samples on ice.

   10. Take out a volume of 60 μL, corresponding to 25 μg, from each of the digested protein samples. Dissolve 800 μg TMTpro 18-plex reagent using 150 μL of water-free acetonitrile, and label each sample with 25 μL of a different TMTpro 18-plex labeling reagent solution. Let the labeling reaction occur at room temperature for 2 h. Keep the remaining 25 μg of each sample at -80 °C as a backup. The remaining TMTpro 18-plex reagent solution can be stored at -80 °C after speed-vacuum drying.

   11. Stop the reaction by adding 50% hydroxylamine solution to quench labeling, to a final concentration of 0.5% hydroxylamine.

   12. Pool the labeled samples together in a 2-mL tube and concentrate the obtained multiplexed sample at least two-fold in a speed-vacuum concentrator.

   13. Acidify the samples using acidifying solution, to a final concentration of ~1% TFA, to reach a final pH < 3.

   14. Perform desalting of the multiplexed sample using a C-18 desalting column and a vacuum manifold. The sample desalting procedure, in sequence, is as follows:

       1. Wash the column with 0.45 mL of 100% methanol.

       2. Wash with 0.45 mL of 50% ACN (v/v) solution including 0.1% TFA (v/v).

       3. Equilibrate with 0.9 mL of 0.1% TFA (v/v); load the sample.

       4. Wash with 0.45 mL of 2% ACN (v/v) solution including 0.1% TFA (v/v).

       5. Wash with 0.15 mL 2% ACN (v/v) solution including 0.1% FA (v/v).

       6. Elute with 0.45 mL of 50% ACN (v/v) solution including 0.1% FA (v/v). Collect the elution in a 2-mL protein low-binding tube.

       7. Elute with 0.3 mL of 80% ACN (v/v) solution including 0.1% FA (v/v). Collect the elution in the same 2-mL protein low-binding tube of the previous step.

   15. Concentrate the desalted and cleaned sample to dryness, using a speed-vacuum concentrator (overnight concentration is possible).

       
       

4. Peptide separation, mass spectrometry, and proteomics data analysis

   1. Resuspend the final multiplexed sample in 100 µL of High pH Buffer A, and use 50 μL (equivalent to 225 μg of original starting protein amount) for the next steps. Keep the remaining at ­80 °C for backup (it is also possible to dry it again in a speed-vacuum concentrator before storing it, which is advised for long term storage).

   2. Inject the resuspended sample for off-line, high pH reversed-phase separation and fractionation into a capillary HPLC system equipped with a 25-cm long, 2.1-mm wide (inner diameter) C18 column, at a flow rate of 200 μL/min.

   3. Perform peptide separation with a binary solvent system consisting of high pH Buffer A and high pH Buffer B, using a gradient from 1% to 6% B from 2 min to 6 min, to 26% B in 36 min, to 53% B in 4 min, to 63% B in 3 min, and then at 63% B for 5 min. Monitor the elution by UV at 214 nm (Figure 1).

      
      

      
      Figure 1. Example of a chromatogram showing the separation of TMT-labeled peptides during high pH reverse phase HPLC, monitored by absorbance at 214 nm (blue), and its gradient (black and dotted line). The light blue area depicts the sample collected in fractions, while the fractions are delimited by light blue and dotted lines.

      
      

   4. Collect 96 fractions of 100 μL in a ninety-six-well plate, starting at the minute 2.5 after injection.

   5. Concatenate the ninety-six 100-μL fractions into 48 concatenated fractions by merging each fraction “n” with “n+48” (for example, 1 with 49, 2 with 50 … and 48 with 96).

   6. Dry the final fractions using a speed-vacuum concentrator.

   7. Inject the equivalent of 1 μg peptides of each fraction on a reversed-phase C18 nano-LC (nLC) column of a nano-LC–ESI–MS/MS instrument, with a C18 nano-trap column and the column for nLC separation at the temperature of 55 °C during peptide analysis.

   8. Perform nLC separation using a binary solvent system, consisting of LC-MS buffer A and LC-MS buffer B, with a gradient of 4% to 26% B from 5 min to 91 min, 26% to 95% B in 9 min, and 5 min in 95% B, prior to equilibration in 4% A (Figure 2).

      
      

      
      Figure 2. Gradient for nano-LC separation of TMT-labeled peptides before MS/MS analysis

      
      

   9. Perform quantitative bottom-up proteomics analysis for TMTpro-based MS/MS acquisition. Acquire mass spectra in a mass-to-charge (m/z) range of 375–1,500, with a resolution of 120 000 at m/z 200 for MS1. Set the AGC target to 3 × 10⁶, with a maximum injection time of 100 ms. Select the 17 most intense peptide peaks for peptide fragmentation via HCD, with the NCE value set at 33. The ion selection abundance threshold is set at 0.1% with charge exclusion of z = 1 ions. The MS/MS spectra are acquired at a resolution of 45 000, with an AGC target value of 2 × 10⁵ ions, or a maximum injection time of 120 ms. The fixed first m/z is 110, and the isolation window is 1.6 m/z. The instrument is operated in the positive ion mode for data-dependent acquisition of MS/MS spectra, with a dynamic exclusion time of previously selected precursor ions of 45 s.

Data analysis

1. Perform identification and quantification of peptides and proteins using MaxQuant or Proteome Discoverer (preferentially with the most recent versions of these software) as proteomic database search engine software. Search against the UniProt complete proteome database accordingly to the species of origin of the used cell culture (e.g., UP000005640 for human samples). When performing the database search for peptide and protein identification, use: cysteine carbamidomethylation as a fixed modification; TMT-related modifications; deamination on methionine oxidation, arginine, and asparagine as variable modifications; trypsin enzyme specificity with maximum of two missed cleavages. Use a 1% false discovery rate as a filter at both protein and peptide levels.

2. Remove all potential contaminants and reversed-hit peptides and include only proteins with at least two unique peptides in the quantitative analysis.

3. For analysis of the obtained protein data, eliminate the proteins with all missing values in all replicates in any treatment and normalize the quantified abundance of each protein in each sample (labeled with a different TMT) over the total intensity of all proteins for that sample.

4. For each protein in each replicate of D1–D5 ligands, divide the normalized protein abundance by the average abundance of this protein in the vehicle-treated replicates. Measure the average ratio across replicates of each D ligand/vehicle control and calculate the Log₂ values of the obtained ratios.

5. Calculate the p-value for the assumption that the above ratio is non-zero using two-tailed Student’s t-test (with equal or unequal variance, depending on F test).

6. For each D ligand, visualize the results by a volcano plot containing the Log₂ of the ratio as x-axis and -Log₁₀ of the corresponding p-value as y-axis.

7. For each D ligand, the target candidates are found among the proteins with large absolute Log₂ of the ratios and simultaneously large -Log₁₀ of the p-values, also in comparison with what was obtained by the other D ligands, with potentially similar or different activities, induced phenotypes, or mechanisms.

Recipes

1. Cell lysis buffer

   Reagent	Final concentration	Amount
   protease inhibitors (100×)	1×	150 μL
   PBS	n/a	1,350 mL
   Total	n/a	15 mL

   
   

2. 20% NP40

   Reagent	Final concentration	Amount
   NP40 (absolute)	20%	3 mL
   Milli-Q water	n/a	12 mL
   Total	n/a	15 mL

   
   

3. 0.5 M DTT solution

   Reagent	Final concentration	Amount
   DTT (absolute)	0.5 M	77 mg
   Milli-Q water	n/a	Add Milli-Q water to dissolve DTT until the volume is 1 mL
   Total	n/a	1 mL

   
   

4. 0.5 M IAA solution

   Reagent	Final concentration	Amount
   IAA (absolute)	0.5 M	93 mg
   Milli-Q water	n/a	Add Milli-Q water to dissolve IAA until the volume is 1 mL
   Total	n/a	1 mL

   
   

5. 20 mM EPPS buffer (pH=8.2)

   Reagent	Final concentration	Amount
   EPPS (absolute)	20 mM	250 mg
   Milli-Q water	n/a	Add 40 mL of Milli-Q water to dissolve EPPS
   10 M NaOH solution	n/a	Adjust the pH until the final pH is 8.2
   Milli-Q water	n/a	Add Milli-Q water until the volume is 50 mL
   Total	n/a	50 mL

   
   

6. 20 mM EPPS buffer (pH=8.2) including 8 M urea

   Reagent	Final concentration	Amount
   Urea (absolute)	8 M	480 mg
   20 mM EPPS buffer (pH=8.2)	20 mM	Add Milli-Q water to dissolve urea until the volume is 1 mL
   Total	n/a	1 mL

   
   

7. 50% ACN solution

   Reagent	Final concentration	Amount
   ACN (absolute)	50% (v/v)	25 mL
   Milli-Q water	n/a	25 mL
   Total	n/a	50 mL

   
   

8. 0.1% TFA (v/v) solution

   Reagent	Final concentration	Amount
   TFA (absolute)	0.1 % (v/v)	50 μL
   Milli-Q water	n/a	Add Milli-Q water until the volume is 50 mL
   Total	n/a	50 mL

   
   

9. 2% ACN (v/v) solution including 0.1% TFA

   Reagent	Final concentration	Amount
   TFA (absolute)	0.1% (v/v)	50 μL
   ACN	2% (v/v)	1 mL
   Milli-Q water	n/a	Add Milli-Q water until the volume is 50 mL
   Total	n/a	50 mL

   
   

10. 2% ACN (v/v) solution including 0.1% FA (LC-MS buffer A)

    Reagent	Final concentration	Amount
    Acetonitrile including 0.1% FA	2%	1 mL
    H2O including 0.1% FA	n/a	49 mL
    Total	n/a	50 mL

    
    

11. 50% acetonitrile (v/v) solution including 0.1% FA

    Reagent	Final concentration	Amount
    Acetonitrile including 0.1% FA	50 %	25 mL
    H2O including 0.1% FA	n/a	25 mL
    Total	n/a	50 mL

    
    

12. 80% acetonitrile (v/v) solution including 0.1% FA

    Reagent	Final concentration	Amount
    Acetonitrile including 0.1% FA	0.5 M	40 mL
    H2O including 0.1% FA	n/a	10 mL
    Total	n/a	50 mL

    
    

13. 20 mM NH₄OH in H₂O (high pH Buffer A)

    Reagent	Final concentration	Amount
    28%–30% NH4OH water solution	20 mM	676 μL
    Milli-Q water	n/a	Add Milli-Q water until the volume is 500 mL
    Total	n/a	500 mL

    
    

14. 20 mM NH₄OH in ACN (high pH Buffer B)

    Reagent	Final concentration	Amount
    28%–30% NH4OH water solution	20 mM	676 μL
    ACN	n/a	Add ACN to dissolve DTT to 500 mL
    Total	n/a	500 mL

    
    

15. 98% ACN in H₂O including 0.1% FA (LC-MS buffer B)

    Reagent	Final concentration	Amount
    Acetonitrile including 0.1% FA	98%	490 mL
    H2O including 0.1% FA	n/a	10 mL
    Total	n/a	500 mL

Acknowledgments

The PISA assay was invented, developed, and further optimized as method development of the Chemical Proteomics Core Facility of the Karolinska Institutet, Stockholm (https://ki.se/en/mbb/chemical-proteomics-core-facility), unique national unit of Chemical Proteomics at the Swedish infrastructures SciLifeLab (Science for Life Laboratrory, https://www.scilifelab.se/units/chemical-proteomics/), and BioMS (Swedish National Infrastructure for Biological Mass Spectrometry, https://bioms.se/technologies/chemical-proteomics/). The Chemical Proteomics Unit provides chemical proteomics support to research projects. SciLifeLab, BioMS, and Karolinska Insitutet supported this work.

  This protocol was adapted from the original proof-of-principle article of the PISA method entitled “Proteome integral solubility alteration: a high-throughput proteomics assay for target deconvolution” (Gaetani et al., 2019).

Competing interests

The authors declare no competing interests.

Ethics

The protocol here described is generally applicable on any cell culture, commonly on commercial cell lines, and does not have any ethics requirement per se. It is to be noted that the protocol is also suitable to be applied to studies on primary cells extracted from human and/or animal tissues, and in such cases the use of cells derived from human or animal sources needs the approval by the ethics committee.

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