TITLE: 702.B.3 URMC HTC Lung Tissue Digestion SOP + Worksheet 052820
AUTHORS: Gloria Pryhuber, Ravi Misra, Tiru Rangasamy, Thomas Mariani, Gautam Bandyopadhyay, Lisa Rogers, Amanda Howell, Claire Wyman, Heidie Huyck, Siva Solleti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)</span></div></div><div class = "text-block">702.B.3 Combined SOP and Worksheet HTC_Lung_Tissue_Digestion_0100616rev061220</div><div class = "text-block"><span style = "font-weight:bold;">Purpose and Scope of the Procedure or Laboratory Assay</span></div><div class = "text-block"><div class = "justify" style = "text-align:justify">1.To dissociate and isolate single cell suspensions from human lung samples to meet the standards and needs of the Human Tissue Core.</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">2.This protocol covers manual gross dissociation of pieces of lung tissue (lobes or cubes) followed by enzymatic digestion and freezer storage as mixed populations of filtered and counted cells for which viability has been determined.</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">3.A separate protocol details the technique of freezing, thawing and further identifying and sorting these mixed cell populations.</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-weight:bold;">Scientific Principles or Validation of Procedure</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>1.Fujino </span><span style = "font-style:italic;">et al. </span><span style = ":UNDERLINE;">Am J Respir Cell Mol Biol.</span><span> 2012 Apr;46(4):422-30</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>2.Barkauskas, C. E., Cronce, M. J., Rackley, C. R., Bowie, E. J., Keene, D. R., Stripp, B. R., ... & Hogan, B. L. (2013). Type 2 alveolar cells are stem cells in adult lung. </span><span style = "font-style:italic;">The Journal of clinical investigation</span><span>, </span><span style = "font-style:italic;">123</span><span>(7), 3025.</span></div><style>
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[STEPS]
?. [Procedure]
Record details of procedure in the Tables at end of this Worksheet. At HTC, the contents of the Worksheet needs to be transferred to BRINDL Database as soon as possible.
?. [Procedure]
Record the time the lung tissue was received for dissociation (see Table 1)
?. [Procedure]
Weigh the total lung tissue received for dissociation (see Table 1)
?. [Procedure]
Record the time the spleen and thymus was received for dissociation (if applicable) (see Table 2)
?. [Procedure]
Weigh the total tissue received of spleen and thymus for dissociation (if applicable) (see Table 2)
?. [Procedure]
Weigh the total lung tissue to be used for dissociation (see Table 1)
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							i)If multiple types of tissue or protocols used, indicate on the Worksheet
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?. [Procedure]
Dissect pleura and larger airways from the lung tissue. To cut out airway +/- pulmonary arteries, follow down the major branches of the bronchi.
?. [Procedure]
Cut the lung tissue into large pieces. Set aside the large airways (bronchi, BRR) for dissociation separate from remaining lung tissue. To ensure an accurate representation of the sample, cut remaining lung tissue into smaller pieces and combine lung tissue from multiple lobes (=CBL, combined lung lobes, if applicable) before dividing lung tissue into C-tubes.
?. [Procedure]
Make enzyme solutions as follows, and prewarm at 37° C just a few minutes prior to use (to ensure optimal enzyme activity)
37 °C
?. [Procedure]
i)Calculating the total number of C-tubes:
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							# C-tubes for lung tissue (max 6 g tissue per C-tube): ________
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							# C-tubes for lung tissue – collagenase only (col only, max 2 g tissue per C-tube): _______
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							# C-tubes for airway (BRR, max 6 g tissue per C-tube): ________
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							# C-tubes for spleen (around 2 g tissue per C-tube) (if applicable): ________ 
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							# C-tubes for thymus (around 2 g tissue per C-tube) (if applicable): ________
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							# C-tubes total: ________ 
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?. [Procedure]
ii)Calculating the total volume of digestion cocktail, buffer and enzymes:
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							    Total Volume of Digestion Cocktail (DC) needed = Total # C-tubes ________ x 10 = ________    Digestion Buffer (DB) = Total Volume of DC needed – Vol of dispase                                             = ___-___ = ____ml DB needed  ABCD1  Enzymes Used
    Weight Added to Buffer (mg)
    Volume Added to Buffer (mL)
    Final Conc. (mg/ml)
  2  Collagenase
    Total vol. DC x 2 = 
    N/A
  2 mg/ml3  DNase
    Total vol. DC x 2 = 
    N/A
  2 mg/ml4  Elastase
    Total vol. DC / 2 = 
    N/A
  0.5 mg/ml5  Dispase
    N/A
    Total vol. DC / 5 = 
  1mg/ml
ABCD1  Enzymes Used
    Weight Added to Buffer (mg)
    Volume Added to Buffer (mL)
    Final Conc. (mg/ml)
  2  Collagenase
    Total vol. DC x 2 = 
    N/A
  2 mg/ml3  DNase
    Total vol. DC x 2 = 
    N/A
  2 mg/ml4  Elastase
    Total vol. DC / 2 = 
    N/A
  0.5 mg/ml5  Dispase
    N/A
    Total vol. DC / 5 = 
  1mg/ml
?. [Procedure]
Re-weigh the lung tissue (CBL) and the airway tissue (BRR) dissociated and record on worksheet (Table 1)
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							i)  CBL weight: ________g
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							ii)  CBL-COL weight: _______g
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							iii) BRR weight: ________g 
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?. [Procedure]
Record weight of spleen dissociated, if applicable: (see Table 2)
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							Record weight of thymus dissociated, if applicable: (see Table 2)
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?. [Procedure]
Using GentleMACS Tissue Dissociator: 
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?. [Procedure]
i)Add no more than 6 g of lung tissue to Miltenyi C tubes containing 10 ml of pre-warmed 1x digestion cocktail (collagenase, dispase, elastase, DNase diluted in digestion buffer). Add no more than 2 g of lung tissue to Miltenyi C tubes containing 10ml of pre-warmed 1x Col Only digestion cocktail (collagenase only). Add no more than 6 g of airway to separate Miltenyi C tube, to be digested under identical conditions as lung tissue, but kept separate from lung tissue throughout processing procedure
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?. [Procedure]
ii)If total weight of tissue exceeds 8 C tubes, exclude some from digest and flash freeze as CBL-BPS, and record weight of biopsy:________g, and time biopsy was frozen: ________
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?. [Procedure]
iii)Cut lung tissue and airway into smaller pieces using scissors in C-tube
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?. [Procedure]
iv)Cut spleen and thymus tissue into smaller pieces using scissors in C tube (if applicable)
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?. [Procedure]
v)Dissociate lung tissue, airway, spleen, and thymus (if applicable) using the GentleMACS mouse tumor implant program 01.01
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?. [Procedure]
vi)Record time tissue added to enzyme and time GentleMACS run ends (See Table 1 and 2)
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							Enzyme__________GentleMACS __________
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?. [Procedure]
vii)After GentleMACS run, loosen caps and place in 37oC 5% CO2 incubator for 60 mins. Invert to mix each tube every 15 minutes (pull stuck stringy stuff off the cap and submerge in enzyme
?. [Procedure]
Dilute the suspension with 5 ml of cold sterile DPBS containing 10% FBS and pass it through a 100 micron cell strainer into a 50 ml conical tube
?. [Procedure]
Disrupt the residual lung fragments in the strainer using a syringe plunger and add 10 ml cold sterile DPBS containing 10% FBS to recover lung cells from the dish / C tube and through the strainer
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							i)Aim for at least 20ml of volume through filter into new 50ml conical. Expect filtering to take as long as 30-45 minutes per tube, depending on age of donor (older donor takes longer)
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?. [Procedure]
Wipe down caps and lips of tubes and place in centrifuge cups which have been equilibrated to 20°C in balanced positions. Attach aerosol containment lids. Pellet cells x10 mins at 1000xg, 4 oC
?. [Procedure]
Gently decant supernatant into waste bucket containing 10% bleach and blot tube lip, watch the pellet to ensure no cell loss
?. [Procedure]
Re-suspend cell pellet from each 50 ml conical tube in 10-30 ml of RBC lysis buffer, depending on redness of cell pellet. Combine all cell pellets, and divide evenly among twice the number of tubes to ensure equal treatment. Incubate for 5 mins at room temperature
?. [Procedure]
Neutralize the reaction by bringing the volume up to 50 ml with cold sterile DPBS containing 10% FBS
?. [Procedure]
Pellet cells for 10 mins at 800xg at 4 oC
?. [Procedure]
Carefully decant supernatant into waste bucket containing 10% bleach (Pellet can still be loose)
?. [Procedure]
Re-suspend the cells in cold DPBS containing 10% FBS (typically 40ml for CBL and 20ml for BRR, SPL, THY, use more volume if pellet is 5ml or greater) for counting cells by trypan blue exclusion method
?. [Procedure]
Pass resuspended cells through 100 micron cell strainer to eliminate debris generated during RBC lysis
?. [Procedure]
Count by hemocytometer: 
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							Dilute 10µL of cells in 90µL 10% FBS in DPBS.Add 100µL 0.4% -4% Trypan Blue solution. 
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							Both large and small cells are counted (="total). % of cells that are relatively large is calculated ("large"). 
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							    Either the total or the large cell count is used for purposes of freezing cells down.
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							Optional: i)Take 50 million cells out for the CBL (full enzyme digestion) and pellet in 15mL conical tube.
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							    Use this cell pellet to freeze first five vials down at 10 million cells per vial 
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?. [Procedure]
Pellet cells for 10 mins at 800xg at 4 oC. Decant supernatant into waste bucket containing 10% bleach
?. [Procedure]
Resuspend cells in appropriate volume of freezing media (90% FBS, 10% DMSO). Aliquot cells in freezing media into cryovials (1 mL per cryovial except last), using a micropipette     i)After the first 5 vials of 10x106 cells/vial, the goal is 40 vials with 40 – 60x106 cells/vial; 
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							            if there is a large yield of cells, freeze down in up to 70 vials
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							        ii)Measure the volume dispensed in last cryovial and record on worksheet table
?. [Procedure]
Label cryovials with appropriate convention: For example: D###-CBL-MIX-## for lung cells, D##-CBL-MIX-COL for lung cells digested with collagenase only, D###-BRR-MIX-## for airway cells, D###-SPL-AMIX for spleen cells, and D###-THY-AMIX for thymus cells
?. [Procedure]
Place cryovials in Mr. Frosty, and freeze at -80 oC    i.). Record time Mr. Frosties are placed in the -80 oC (Freeze time Table 1 and 2)
?. [Procedure]
After at least 24 hours, and as soon thereafter as possible, transfer cells to liquid nitrogen tank for long-term storage. Enter vials and location into BRINDL Database (if applicable)
?. [Analysis of Results :]
Use Tables below or Worksheet (702.A.X) and BRINDL Database CRF to Record the following:a.Personnel Involved in Isolation
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							b.Lung Lobe or Block(s) Used for Dissociation
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							c.Weight of Tissue Received for Dissociation 
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							d.Weight of Tissue Used for Dissociation
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							e.Method of Mechanical Dissection Used
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							f.Enzymes and concentrations used in digestion
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							g.Calculate and Record:
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							    i)Total Cell Number Yield 
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							    ii)Total Large Cell Number Yield 
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							    iii)% Viability 
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							    iv)% of Total Large Cell Yield 
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							    v)Number Viable Cells
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							    vi)Total Cell per gram of tissue 
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							    vii)Total Large Cell per gram of tissue 
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							    viii)Numbers of Vials Frozen 
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							        (1)# Cells per Vial 
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							            (2)Volume of Last Vial
?. [Table 1.]
?. [Table 2.]