Production of CD4-LV and CD8-LV
Seeding of Lenti-X 293T cells
Timing: Day 1 early afternoon
1) On the day prior to transfection, seed 2x107 cells in 18 ml DMEM (10% FBS, 2 mM L‑glutamine) per 15 cm cell culture plate (growth surface 147.8 cm2) in 40 plates to achieve 75-90% cell confluency for transfection. For that, add 8 ml medium to the culture plates, then dilute the cells to a concentration of 2x106 cells/ml and add 10 ml of this cell suspension to the plates.
Note: To have enough cells for the production expand your cells to ~18 T175 flasks four days before seeding. Check the condition of the cells before seeding. For the transfection, we prefer to work with 15 cm culture plates instead of T175 flasks, though this is also possible. Plates have a higher risk for contaminations, but allow a quicker workflow as they are easier to handle, especially when many plates have to be processed.
Δ CRITICAL Handling of the plates: Be careful not to move your hand or material above an uncovered plate to avoid contaminations. Evenly spread the cells on the plates by carefully tilting the plate back and forth and from left to right after seeding.
Transfection of Lenti-X 293T cells Timing: Day 2 late afternoon
2) Observe cells under the microscope to check for their condition and possible contaminations. The cells should be equally distributed over the plate (no cluster formation) with a confluency of around 75-90% and should not grow 3-dimensionally.
Δ CRITICAL The colour of the medium should be orange, not red (low cell density) nor turbid yellow indicating a contamination.
The cells must at least cover 75% of the plate’s surface to transfect them. They should rather be above this threshold, yet they have to survive two more days in culture.
3) Replace medium in all dishes by 12 ml DMEM containing 15% FBS and 2 mM L‑glutamine
Δ CRITICAL Cells should not be kept with this low amount of medium for longer than 1 h 30 min. This can be achieved by first changing the medium of 20 plates, then starting to prepare the DNA Mix and PEI Mix (step 4), and performing the medium change of the remaining 20 plates during the incubation time of the transfection mix.
4) Separately prepare the DNA Mix and PEI Mix required for transfection in conical-bottom, sterile polypropylene tubes. First, prepare DNA Mix in a 250 ml tube and then prepare PEI Mix in another 250 ml tube. The following amounts are for transfection of 40 cell culture plates:
· DNA mix: Combine 92 ml DMEM without any supplement with 606.9 µg pS‑CD19.CAR‑W and 577.8 µg pCMV‑dR8.91. As a next step, add 35.9 µg of pCAGGS-NiV-GcΔ34-αCD8opt and 179.5 µg pCAGGS-NiV-FcΔ22 for production of CD8-LV or 53.9 µg of pCG-Hmut-CD4.DARPin29.2 and 161.5 µg of pCG-FcΔ30 for production of CD4-LV.
· PEI mix: Combine 5.6 ml PEI with 88 ml DMEM without supplements
Note: Amounts can be downscaled to e.g. five 15 cm cell culture plates per vector.
The total amount of plasmid DNA per plate is 35 µg with a ratio of 1:3:11.3:10.7 between targeted glycoprotein plasmid, fusion protein plasmid, transgene plasmid and packaging plasmid for CD8-LV (as for other NiV glycoprotein based LVs) and a ratio of 1:5:16.9:16.1 for CD4-LV (as for other MV glycoprotein based LVs). The amount of PEI per µg of DNA is 3.68 µg.
Note: The transgene plasmid can be freely chosen. When setting up LVs targeted to other receptors we recommend using GFP for validation. Plasmid ratios may have to be adapted when using transgenes of different size. For third generation packaging LVs plasmid amounts have to be adapted as follows: Instead of using 577.8 µg pCMV-dR8.91 use 385.0 µg of the plasmid coding for Gag/Pol (pMDLg/pRRE, addgene #12251) and 192.77 µg of the plasmid coding for Rev (pRSV-Rev, addgene #12253). The other plasmid amounts are not changed.
Note: VSV-LVs should be produced as a positive control, when testing new transgenes. Use 245 µg of the VSV-G envelope plasmid (pMD2.G, addgene #12259), 700 µg of the transgene plasmid and 455 µg of the packaging plasmid.
5) Thoroughly mix PEI-Mix and DNA-Mix individually by vortexing for 30 seconds, respectively. Then add PEI-Mix to DNA-Mix and vortex again for 30 seconds. Incubate for 20-30 min at room temperature.
Δ CRITICAL To not exceed this incubation time medium change of at least 20 plates should be completed before preparing the transfection mix.
6) After the incubation period, pipette 4.6 ml of the transfection mix from step 5 to each plate using a 10-ml pipette. Carefully tilt the plates to disperse the medium evenly.
Δ CRITICAL Avoid disturbing the attached cells by slightly tilting the plate and carefully pipetting the solution SLOWLY to its border. Distribution of the transfection mix drop by drop is not necessary. Transfect only 10 plates at a time and leave the rest of the plates in the incubator to keep the cells in optimal culture condition.
7) Return the dishes to the tissue culture incubator with a humidified atmosphere containing 5% CO2 at 37°C and incubate them over night for 16-18 h.
Δ CRITICAL Stack the dishes horizontally to make sure cells are completely covered with medium any time.
Note: Transfection can also be performed in the morning (if cell density is > 75%). In this case, medium is replaced 6-8 h after transfection on the same day. Harvest of LV particles will still be two days after transfection, as described in this protocol.
Medium change Timing: Day 3 early morning (~16 h post transfection)
8) Gently aspirate the medium from the cells and add 18 ml DMEM (10% FBS, 2 mM L‑glutamine). Incubate the plates in a tissue culture incubator with a humidified atmosphere containing 5% CO2 at 37°C for 24 h.
Δ CRITICAL Only take 10 plates at a time out of the incubator to keep the cells in optimal culture condition. Change the medium of only two plates at once to avoid cell dehydration.
Note: If cells were transfected in the morning, medium is replaced 6-8 h after transfection on the same day. When using T175 flasks 20 ml DMEM is required.
Harvest of lentiviral vector particles Timing: Day 4 morning
9) To get rid of cell debris, filter the cell supernatant through a 0.45 µm Bottle Top Filter placed on a 500 ml storage bottle. One filter is sufficient for filtration of the supernatant of 20 plates. Filter the LV-containing supernatant by connecting a pump to the filter and collect the flow through.
Δ CRITICAL Keep the supernatant on ice after filtration.
PAUSE POINT Store at least one aliquot of the unconcentrated filtered cell culture supernatant at ‑80°C (4°C in case of titration on the next day) to later compare titers of unconcentrated and concentrated vector stocks. An additional vector harvest after another 24 h can be considered to increase vector yields.
Concentration of vector stock over sucrose cushion Timing: Day 4 – 5
10) Distribute the filtered supernatant to four 250 ml conical centrifuge tubes. Aspirate 14 ml of the 20% sucrose solution with a 10-ml pipette. Insert the pipette to the bottom of the centrifuge tube containing the filtered supernatant and slowly underlay with 9 ml of the sucrose solution. Stop expelling the solution when it hits the 5 ml mark on the pipette. Discard the rest of the solution and use a new pipette for the next tube.
Note: The extra volume in the pipet is necessary to avoid bubbles in the tube, since the 5 ml mark of a 10-ml pipet is still visible when being inserted in the centrifuge tube containing the supernatant.
Δ CRITICAL The sucrose cushion should be pipetted very gently to avoid mixing of the two phases.
11) Balance the tubes by addition of PBS w/o Mg2+/Ca2+ until the weight difference is below 0.1 g.
Note: Weighing can be done outside the cell culture hood, although the tubes have to be kept closed. However, PBS has to be added under the cell culture hood to maintain sterility.
12) Centrifuge for 24 h at 4°C (4500×g; acceleration: 6; deceleration: 6) in a benchtop centrifuge.
Δ CRITICAL The centrifugation time should be set for at least 24 h or more. Set the centrifuge on “Hold” to be sure it does not finish before you are ready to proceed.
Note: You can also concentrate the vectors by ultracentrifugation (SW32 Ti Rotor) for 2 h at 100,000×g, but due to the volume restrictions, we prefer low speed centrifugation. If you decide for ultracentrifugation, we recommend using Open-Top Thinwall polypropylene tubes (Beckmann Coulter, 38.5 ml, 25x89 mm, cat. no. 326823) for centrifugation of the supernatant. These tubes have to be filled to the maximum to prevent collapsing. If necessary, fill up the supernatant to exactly 30 ml with PBS w/o Mg2+/Ca2+ before adding 5 ml sucrose solution.
Note: If production is downscaled to less than 10 plates per vector, concentration of LVs can be performed in 50 ml conical centrifuge tubes. In this case, supernatant should be aspirated with a 10 ml pipette and filtered with a 0.45 µm syringe filter. One filter can be sufficient for two plates by carefully detaching the pipet without clogging the filter (e.g. by keeping ~ 3 ml volume in the pipet). The filtered supernatant of two culture plates is transferred in a 50 ml centrifuge tube and underlayed with 4.5 ml 20% sucrose by aspirating 7.5 ml of the sucrose solution with a 5-ml pipette and stopping to expel the solution when it hits the 3 ml mark on the pipette.
Resuspension of concentrated lentiviral vectors Timing: Day 5
13) Carefully remove the tubes from the centrifuge and discard the supernatant. Remove residual liquid by leaving the tube for 5 minutes upside down in a rack lined with paper towels. Wipe the tubes with fresh paper towels without touching the pellet. Change gloves after that step. A tiny, beige-brownish pellet should be visible at the bottom of the tube.
Δ CRITICAL Pour the supernatant quickly but carefully and avoid bubbles.
14) Add 600-1000 µl PBS w/o Mg2+/Ca2+ to each pellet (equals 60-100 µl per plate) without touching it and let the pellet resuspend for 30 minutes by placing the tubes on ice on a plate shaker. Pipette up and down 80 times with an electronic pipette. Pool the liquids from all resuspended pellets in one of the conical tubes and aliquot the vector stock in low binding tubes in volumes of 15-200 µl (15-25 µl for nanoparticle tracking analysis (NTA) or p24-ELISA and titration, 200 µl for in vivo analyses). Store the vectors at ‑80°C.
Note: Especially for LVs containing MV envelope proteins resuspension of the pellet in a too low volume can be counterproductive. We recommend addition of 100 µl PBS per plate, when starting with a new type of targeted LV.
Note: It is also possible to use a conventional mechanical pipette for resuspension of the pellet.
Δ CRITICAL Avoid foam formation during resuspension of the pellets to prevent aerosols.
PAUSE POINT Lentiviral vectors can be stored at -80°C. If you want to proceed directly with the titration, be sure to freeze/thaw the respective aliquot first to make it comparable with LVs used after storage, e.g. for in vivo applications.
Quantification of particle number and gene transfer activity of lentiviral vectors
A) Determination of the particle number via NTA:
15) Particle numbers are determined via NTA with Nanosight NS300 according to the user’s manual. LVs should be diluted 1:1,000 - 1:10,000 in PBS w/o Mg2+/Ca2+ (typically 1:3,000) in a total volume of 1 ml for measurement (Fig. 4b).
Note: Particle numbers of LVs can also be measured via p24 ELISA according to the manufacturer’s protocol.
B) Gene transfer activity on target receptor positive cells
16) To determine the gene transfer activity of the produced vector particles, CD8+/CD4+ cells are incubated with 5 µl vector stock (consecutive dilution of 1/5) per 4x104 cells in 96-wells. For this purpose, cells are seeded in 100 µl RPMI medium (10% FBS, 2 mM L‑glutamine) and the vector stock, diluted in 100 µl medium, is added to get a final volume of 200 µl per well. The percentage of transduced cells is determined via flow cytometry three or four days later. Antibodies depend on the corresponding transgene. In this case, an anti-myc-PE antibody was used for detection of the CAR (Fig. 4c).
Note: If other targeting vectors are produced, the cell line for titration and the used antibody for detection of the transgene must be adjusted. Vectors should have around 1x107 t.u./ml or above (Fig. 4d).
17) It is also very important to check if the LVs are able to successfully transduce primary cells. For this purpose, human PBMC are purified from whole blood or buffy coats from healthy donors via a Ficoll gradient (e.g. Histopaque 1077, Sigma Aldrich, cat. no. 10771) as described in the manufacturer’s protocol.
For the activation of 2x106 PBMC, a 24-well is coated with 500 µl of 1 µg/ml anti-human CD3 mAb (clone: OKT3, Miltenyi Biotec) and incubated for 2 h at 37°C or overnight at 4°C. Remove medium and replace with 2% BSA in PBS w/o Mg2+/Ca2+ (sterile filtered) for 30 min at 37°C. After washing with PBS w/o Mg2+/Ca2+ twice, 2x106 PBMC are seeded in 2 ml T Cell medium (TCM) supplemented with 3 µg/ml anti-human CD28 mAb (clone: 15E8, Miltenyi Biotec) and 50 U/ml IL‑2 and the plate is incubated for 72 h at 37°C, 5% CO2 and 90% humidity. 0.4-1x105 activated PBMC are seeded per 96‑well in a volume of 100 µl in TCM + 50 U/ml IL‑2. PBMC are then transduced with 5 µl vector stock diluted in 100 µl medium per well. Spinfection is performed at 850×g (acceleration: 7, deceleration: 7) for 90 minutes in a preheated centrifuge (32°C). Afterwards, 100 µl TCM are added into each well and cells are incubated at 37°C, 5% CO2 and 90% humidity. After a cultivation of 4‑7 days, the number of transduced cells is determined via flow cytometry with the corresponding antibodies.
Note: PBMC activation can be adjusted according to the amount of cells needed. E.g. you can activate 1x107 PBMC in a 6-well using 1 ml anti-human CD3 mAb (1 µg/ml) for coating, and seeding cells in a total volume of 6 ml per well.
Note: The amount of vector stock per well when transducing primary cells might have to be adjusted according to the gene transfer activity of the vectors determined on Molt 4.8 cells. We use 5 µl of LVs with titers of 1-5x107 t.u./ml.
Note: Vectofusin‑1 can be used to enhance gene delivery.
Humanization of mice
Mice conditioning with sublethal injection of busulfex TIMING 1 h for 20 mice
Δ CRITICAL Busulfex injection has to be performed 30-36 h prior cell injection
18) Weigh each NSG mouse and calculate the precise volume of busulfex to apply a dose of 20 mg/kg per mouse
Note: stock solution of busulfex (6 mg/ml) can be pre-diluted 2-fold with sterile PBS just before injection.
19) Carefully remove the animal from the cage and restrain it gently in the head‐down position.
20) Insert the needle with the bevel facing ”up” into the lower right quadrant of the abdomen towards the head at a 30‐40° angle to the horizontal line.
21) Inject the appropriate volume of busulfex solution intra-peritoneally in a steady, fluid motion with a 29G 0.5 ml syringe
Note: Busulfex solution for injection is very viscous, therefore slow injection is recommended.
22) Repeat Steps 18 to 21 until the last mouse has been injected. Place the young adult mice back in their box.
Cell preparation for human CD34+ stem and progenitor cells (HSPC) before injection TIMING 40-50 min
Note: HSPCs from three to four donors can be pooled to obtain enough cells to humanize a larger cohort of mice with homogeneous engraftment levels.
Δ CRITICAL It is crucial to use hCD34 cells with a purity that is higher than 90% to obtain a high level of human cell engraftment in NSG mice. A contamination of T cells (> 3%) might result in low human cell engraftment.
23) Warm up 30 ml CellGro medium in a 37°C water bath.
24) Take cells from the liquid nitrogen tank and thaw them in the water bath at 37°C for 1-2 min. Cells should be taken out of the water bath when they are almost thawed.
25) Transfer CD34+ cells very quickly to a prewarmed 50 ml tube containing 30 ml CellGro medium.
26) Spin the tube at 300×g for 10 min at room temperature.
27) Aspirate the supernatant and resuspend cells in 2 ml CellGro medium.
28) Count the cells using a hemocytometer after trypan blue staining (mix 10 μl cells with 10 μl trypan blue and count clear cells as live cells and blue cells as dead cells).
29) Spin down the cell suspension at 300×g for 10 min at 4°C and discard the supernatant.
30) Resuspend the cells in 1 ml of complete CellGro medium at a density of 1-106 living cells/ml and seed in a 24-well plate.
31) Incubate cells at 37°C, 5% CO2, 20% O2 overnight (16 h to 24 h).
Cell preparation for injection TIMING 1h
32) Prior to transplantation, collect cells in 1.5 ml tubes.
33) Count cells using a hemocytometer after trypan blue staining (see step 28).
34) Spin down CD34+ HSPCs at 300×g for 10 min at RT and resuspend them with sterile PBS w/o Mg2+/Ca2+ to 0.7x105 - 2x105 cells per 35 μl total volume for each mouse to be injected.
Δ CRITICAL Cell suspension must be prepared in 35 μl PBS for retro-orbital injection.
Note: The dose of CD34+ cells injected will determine the speed of NSG humanization. If one injects 2×105 CD34+ cells per mouse, 40% human cell reconstitution in blood will be achieved 12-16 weeks post-engraftment.
Transplantation of human cells TIMING 1 h (for 20 mice)
Δ CRITICAL To perform this part of the protocol dedicated personnel trained for animal experimentation is required.
35) Take the cells from Step 34 to the animal facility (experimental area).
36) Mix the cells immediately prior to injection each time by pipetting up and down. Load the syringe (29‑gauge needles, 0.3 to 0.5 ml insulin syringes) with the cell suspension at a volume of 30 µl per mouse. The same syringe can be used for several mice.
Δ CRITICAL Air bubbles in the syringe must absolutely be avoided before injection into the blood stream, since this will lead to cardiac arrest.
37) Mice should be anaesthetised because the needle is being placed in the retro-bulbar space.
Note: Preferentially use inhalant anaesthetic, because it assures rapid induction and quick recovery times.
Δ CRITICAL Make sure to perform anaesthesia and retro-orbital injection under a laminar flow (PSM2, sterile conditions). Decontaminate the surface of the laminar flow and your gloves regularly with 70% alcohol before touching the NSG mice.
38) Induction of anaesthesia is achieved using a decontaminated induction box infused with 3.5% isoflurane enriched air until the mouse is non-responsive, recumbent, and demonstrates a slower, even respiratory pattern. Anaesthesia is maintained using a nose cone releasing 1‑2% isoflurane enriched air.
Note: Make sure the mouse is completely under anaesthesia before injecting the cells. This can be checked by pressing the foot pad to make sure the mouse has no reflexes.
39) Remove the mouse from the isoflurane chamber and place it on a sterile compress with its belly facing the surface of the laminar flow.
40) The mouse’s right eyeball is protruded from the eye socket by applying gentle pressure to the skin at the dorsal and ventral part of the eye.
41) Introduce the needle bevel down at an angle of approximately 30° into the medial canthus.
42) Inject the cell suspension slowly and smoothly and remove the needle slowly and smoothly once the injection is complete.
Note: There should be little or no bleeding.
Note: i.v. injection takes less than 15 sec per mouse. Mice can be maintained under anaesthesia using a nose cone and 1‑2% isoflurane, but injection can also be performed directly after withdrawing the mouse from the induction box if personnel is trained to perform it quickly.
43) Place mice back into the cage for recovery. A warming device is not required, because the injection procedure takes only a very short time (less than 15 sec). Thus, the mouse is usually ambulatory within 30‑45 sec.
Note: i.v. injection can also be performed into the tail vein. This has the advantage that there is no need for anaesthesia of the mice, but requires an appropriate “contention box”, as also a mouse-tail illuminator for dilatation of the tail vein before injection.
Note: Perform the equivalent procedure as stated above for the control mice, but instead of HSPC inject a vehicle using a buffer without cells or use mice that are not transplanted.
Δ CRITICAL The NSG mice are immunodeficient and should be housed in a sterile environment. They must be strictly handled under a laminar flow, get sterile food and water and are kept in sterile cages. This is essential to ensure an efficient high-level humanization of these mice. If this is not respected, opportunistic infections of NSG mice will strongly impair hCD34+ cell engraftment.
Determination of the humanization level in the peripheral blood (PB) TIMING 2-3 h
Note: The complete humanization process can take 12 to 16 weeks depending on the hCD34+ cell donor and the number of injected cells. Eight weeks after human cell injection, blood is taken from the mice every 3 weeks to monitor humanization in the peripheral blood.
44) Apply a drop of tetracaine 5 min prior to sampling of blood from the eye.
45) Perform retro-orbital blood sampling by penetrating the retro-orbital sinus in mice with a sterile haematocrit capillary tube or Pasteur pipette.
Note: Sterile tubes or pipettes are required to avoid periorbital infections and potential long-term damage to the eye. The eyelid is pulled back to protrude the eye and facilitate blood harvesting.
46) Take approximately 100 µl blood per mouse in 1.5-ml sterile microcentrifuge tubes containing 20 µl of CPD (for plasma collection and gDNA analysis) and immediately pipet 50 µl in a new tube containing 20 µl of CPD and place on ice for FACS staining.
Note: Samples can be kept on ice for up to 12 h.
Note: Mice can be briefly anaesthetised with isoflurane (Step 38) to facilitate blood sampling.
Determination of humanization level in PB by flow cytometry:
47) Add 50‑100 µl PBS w/o Mg2+/Ca2+ to the blood (1/1 ratio).
48) Transfer 50 µl of 2x-diluted blood into 5 ml FACS tubes containing 50 µl of FACS Wash Buffer supplemented with the antibody cocktail described in panel 1 (Table 1).
49) Incubate for 30 min at 4°C in the dark.
50) Add 500 µl FACS Wash Buffer.
51) Centrifuge at 300×g for 10 min at 4°C.
52) Discard supernatant by inverting tube and gently tapping on paper towel to remove the remaining drop of supernatant.
53) Add 700 µl of 1X RBC lysis buffer for 10 min at room temperature (dark).
Note: Depending on the RBC lysis composition, incubation time can vary (2 - 10 min.)
54) Centrifuge at 300×g for 10 min at 4°C, discard supernatant and add 3-4 ml FACS Wash Buffer.
55) Spin the cells again at 300×g for 10 min at 4°C and remove supernatant.
56) Resuspend the cell pellet in 100-200 µl FACS Fixation Buffer
57) Keep in FACS tubes and then analyse via MACS Quant measurement.
Note: RBC lysis and staining can also be performed directly in 96 well plate (conical bottom). Adapt volume and use 200 µl of 3X RBC lysis buffer.
58) The white blood cells are gated by granularity and size (FSC vs. SSC), and are then evaluated by gating for hCD45 (negative for mCD45) combined with hCD3 (total T-cells), hCD19 (total B-cells), or hCD14 (monocytes) using flow cytometry analysis (Figure 5).
59) Determine the human immune reconstitution by calculating the following formula
Human immune reconstitution = % human CD45 cells / (% human CD45 cells + % murine CD45 cells)
IL7 conditioning TIMING 45 min (for 20 mice)
Δ CRITICAL Human IL7 needs to be injected four days and again one day before vector application.
60) Resuspend human IL7 according to manufacturer’s protocol.
61) Prepare a working solution at 2 µg/ml in sterile PBS w/o Mg2+/Ca2+.
62) Inject 100 µl of the working solution of huIL7 (200 ng) via the sub-cutaneous route.
Restrain the animal by grasping the skin along its back and insert the needle at the base of the skin fold between thumb and finger. Administer IL7 in a steady, fluent motion with a 29G 0.5 ml syringe.
Note: The IL7 stock solution is at 100 µg/ml.
Note: The control group receives PBS using the same route of administration.
In vivo administration of the vector TIMING 1 h
63) Inject a single dose of 2×1011 LV particles i.v. using the same procedure as for human CD34+ cell injection (step 36-43).
Δ CRITICAL A maximal volume of 100 µl can be injected into the eye.
Note: Alternatively, up to 200 µl can be injected into the tail vein.
CAR-T longitudinal analysis TIMING 1-8 weeks
Note: For longitudinal analysis, sample blood from the mice as described in step 44-46 every week following the first IL7 injection.
64) Monitor mice every 3-4 days and euthanize them at the chosen time point after LV injection or when clinical endpoints are reached.
Δ CRITICAL Define appropriate experimental endpoints (physical appearance, behavioural changes, weight loss) according to the protocol that was submitted at the local ethical committee.
Organ collection TIMING 2 h for 20 mice
Δ CRITICAL At least two trained people are necessary for mouse euthanasia and organ collection.
65) Anaesthesia of the mice is done by i.p. administration of 100 mg/kg ketamine / 20 mg/kg xylasin.
66) Collect >500 µl of blood in a 1.5 ml tube containing 100 µl CPD via retro-orbital blood sampling or intracardiac puncture.
67) Perform cervical dislocation.
68) Collect mouse tissues (spleen, lymph nodes, liver and others) in FACS Wash Buffer and keep them on ice immediately.
PAUSE POINT Organs can be stored at 4°C ON before cell isolation.
69) For histology and immunohistochemistry fix the tissue in formalin 4% (for 24 h) and wash in 70% ethanol. Keep the organs in alcohol until paraffin-embedded slices are prepared from the fixed tissues.
Cell isolation from haematopoietic tissues TIMING 4 h
Δ CRITICAL At least two trained people are necessary for organ and blood processing
Sampling of peripheral blood at final analysis:
70) Blood collection is performed as described in step 44-46. For final analysis 400-600 µl blood are used per mouse.
Note: At time points before final analysis, blood is collected as described in step 44-46.
Note: Put 20 µl of blood into a 1.5 ml tube and store at -80°C for gDNA extraction and qPCR (step 106-111).
71) Centrifuge blood at 300×g for 5 min at room temperature.
72) For plasma collection, transfer the upper phase to a 1.5 ml tube and centrifuge at 14,000×g for 5 min at 4°C. Transfer the supernatant to a fresh 1.5 ml tube and store at -80°C.
73) Wash the remaining blood cells with 1 ml PBS w/o Mg2+/Ca2+ and centrifuge at 300×g for 5 min at 4°C.
74) Add 700 µl of 1X RBC lysis buffer for 10 min at room temperature (dark).
Note: Depending on the RBC lysis composition, incubation time can vary (2 - 10 min.)
75) Perform the washing step as indicated in step 73.
76) Resuspend the pellet in 1 ml PBS w/o Mg2+/Ca2+ and count cells with trypan blue solution (step 28).
77) Transfer the single cell suspension to micronics for flow cytometry analysis (steps 98-105).
Mononuclear cell isolation from spleen:
78) Place a 40 µm cell strainer on top of a 50 ml Falcon tube.
79) Put the spleen on the filter and use the plunger base of a syringe to mash the spleen on the filter while pouring PBS w/o Mg2+/Ca2+ through the strainer until spleen is completely dissociated.
80) Centrifuge at 300×g for 10 min at 4°C.
81) Discard supernatant and resuspend the pellet in 3 ml PBS w/o Mg2+/Ca2+.
82) Put 2 ml Lymphoprep solution in a 15 ml tube.
83) Slowly add 3 ml cell suspension on top of the Lymphoprep.
Δ CRITICAL Gently pipet the cells to avoid mixing the two phases.
84) Centrifuge at 850×g for 20 min at 20°C (acceleration: 1; deceleration: 0).
85) Carefully aspirate the upper phase leaving 2-3 ml above the ring of mononuclear cells.
86) Carefully aspirate the ring of mononuclear cells with a 10 ml pipette and put into a new 50 ml tube.
87) Fill up the tube with PBS w/o Mg2+/Ca2+.
88) Spin at 300×g for 10 min at 4°C.
89) Resuspend the pellet with 5-10 ml PBS w/o Mg2+/Ca2+ to reach 1-5x106 cells/ml.
90) Count cells with trypan blue solution (step 28).
Note: Cell suspension should be diluted 20-fold with PBS for cell counting in a 96-well plate.
Note: Alternatively, resuspend the cell pellet with 1 ml RBC lysis buffer instead of doing a cell isolation on the lymphoprep gradient. Incubate for 5 min at room temperature and dilute the lysis buffer by adding 5 ml PBS w/o Mg2+/Ca2+ before centrifugation.
91) Use the single cell suspension from spleen for DNA extraction (step 106) and flow cytometry analysis (steps 98-105): Put 1x106 cells in a FACS tube and 2x106 cells in 1.5 ml tube for qPCR for further analysis. The remaining cells are frozen at -80°C in freezing medium.
Mononuclear cell isolation from the bone marrow:
92) Put the femur into a 10 cm dish.
Note: Tibia can also be processed using this procedure.
93) Remove the muscles and residual tissue surrounding the femur with sterile forceps and scissors.
94) Cut the femurs at both ends with sharp sterile scissors. Use a 23- or 25-gauge needle and a 10 cc syringe filled with ice-cold PBS w/o Mg2+/Ca2+ to flush the bone marrow onto a 40 µm nylon cell strainer placed in a 50 ml Falcon conical tube until the flow through turns transparent.
95) Dissociate the bone marrow through the cell strainer with a 5 ml plunger and wash the strainer with PBS w/o Mg2+/Ca2+.
96) Centrifuge cells at 300×g for 10 min at 4 °C. Discard the supernatant.
Note: Optional: Resuspend the cell pellet with 1 ml RBC lysis buffer. Incubate for 5 min at room temperature and dilute the lysis buffer by adding 5 ml buffer before centrifugation.
97) Resuspend the pellet with 2 ml PBS w/o Mg2+/Ca2+, count bone marrow cells with a hemocytometer with trypan blue (step 28) and adjust concentration for further analysis as for spleen cells.
Analysis of in vivo samples
A) FACS based phenotypic characterization of in vivo generated CAR T cells TIMING 2 h for staining, 3 h for measurement
Δ CRITICAL Use antibody panel 2 (Table 2) for the following steps
98) Single cell suspensions from spleen, bone marrow and blood from each mouse are prepared and counted. 1x106 cells from each organ are placed into separate micronics and are washed twice with 500 µl FACS Wash Buffer. Pellet cells at 400×g for 5 min at 4°C.
Note: Fluorophores can be exchanged dependent on the laser configuration of the flow cytometer used. The panel can be extended e.g. with anti-human CD20 (LT20), anti-human CD69 (FN50), anti-human CD71 (AC102) and anti-human TIM3 (F38-2E2).
99) Cell pellet is resuspended with blocking solution (tube 1) and incubated for 10 min at 4°C.
100) Add 50 µl of prepared antibody mixture (tube 2) or isotype control mixture (tube 3) into the respective well and incubate for 30 min in the dark at 4°C.
Δ CRITICAL Fluorescent minus one (FMO) controls should be included in case of a spread/spillover of one channel into the other. FMO controls enable to remove all ambiguity from the compensated plots and help to distinguish false positive from actual positive signals.
Note: Ultracomp eBeads are used for compensation. Add 1 µl of each monoclonal antibody to a drop of the compensation beads in micronics.
Δ CRITICAL Vortex the beads prior to use.
Note: Naïve and stem cell subsets may be additionally identified by including CD45RO. In this case, naïve T cells are identified as CD45RA+CD62L+CD45RO- whereas stem cells are CD45RA+CD62L+CD45RO+.
101) Wash cells with 500 µl FACS Wash Buffer. Centrifuge at 400×g for 5 min at 4°C. Discard the supernatant carefully and repeat this procedure.
102) Add 100 µl of viability dye (tube 4) to each well and incubate for 20 min in the dark at 4°C.
103) Perform the washing step as indicated in step 101.
104) Add 100 µl of FACS Fixation Buffer and resuspend the pellet.
105) Proceed to sample acquisition by flow cytometry and analysis by FlowJo software (Fig. 6).
B) Quantitative polymerase chain reaction (qPCR) to determine the number of vector copies associated with genomic DNA extracted from huNSG mouse tissues.
TIMING Enrichment of cells: 2 h, Isolation of gDNA: 3-4 h, qPCR: 3 h
Note: We use WPRE to quantify CAR gene transfer and human albumin as a housekeeping gene. Alternatively, other primers and target sequences can be used.
106) Enrich 2×106 cells from bone marrow or spleen for CD4+ and CD8+ cell population according to the manufacturer’s protocol, respectively and then freeze at -80°C as a pellet for further analysis.
Δ CRITICAL Start with 2×106 cells from each bone marrow or spleen sample as a starting material before enrichment to obtain around 1×105 cells for DNA isolation. Perform the enrichment before freezing the cells.
Δ CRITICAL Enrichment of the target cell population for CD4-LV or CD8-LV, respectively, can be used to confirm the selectivity of the vector for the CD8+ or the CD4+ population by qPCR.
107) Isolate high-molecular weight genomic DNA from the enriched cells using the DNeasy kit, according to the manufacturer’s instructions. Elute DNA with 200 µl of elution buffer and determine DNA concentration using Nanodrop.
108) 12.5 µl of LightCycler 480 Probes Mastermix are mixed with 0.2 µM of each primer and 0.2 µM of each probe in a total volume of 15 µl per sample.
Note: Dilute with DNase- and RNase-free water at all steps.
109) 15 µl of the reaction mix are transferred into the wells of a 96-well PCR plate, then 10 µl of sample containing 100 ng of DNA are added to the plate without mixing to achieve a total volume of 25 µl. As a control, 10 µl of serial dilutions (from 1x106 – 1x100 molecules/µl) are transferred to the plate containing the reaction mix. As a negative control, 10 µl of DNase and RNase-free water are added to the plate.
Note: All samples and controls should be measured in duplicates or better triplicates.
110) The plate is sealed and centrifuged at 20×g for 1 min at 4°C to mix samples with reaction mix.
Δ CRITICAL When using a volume of 10 µl of the standard plasmid with a concentration of 1x106 molecules/µl per well that means you have added 1x107 molecules in total to your sample. This has to be taken into account during analysis with the LightCycler 480 software, where you have to provide the total amount of standard.
111) qPCR is then performed with the LightCycler 480 Instrument II (5 min at 95°C, 45 cycles of 10 sec at 95°C and 40 sec at 60°C). A FAM-labelled probe is used to detect albumin (excitation 465 nm, emission 510 nm). A probe with a Cy5-fluorophore enables CAR detection via WPRE (excitation 618 nm, emission 660 nm). Data are analysed with the LightCycler 480 software using absolute quantification.