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1. Cert_Auth_Ask: CertVLR, R1 2. Cert_Auth_resp: (Ks)KUVLR, [CertMS||(R2||R1)KRMS]Ks 3. Cert_Auth_Ack: (Ks)KUVLR, [Ticket||(R1||R2)KRVLR]Ks where the session key is R1 R2. If the MS stays at the same cell and requests the service several times, we use the TBA protocol. The TBA protocol uses the ticket in the authentication procedure and is described as follows: 1. Ticket_Auth_Com: TID, (Ticket||R1)K0 2. Ticket_Auth_Result: (R2||R1)K0) where the session key is R1 R2. If the ticket is out of date, the MS runs the CBA protocol to get the current ticket. TBA protocol uses a symmetric crypto-system in authenticating the MS. Therefore, the computational cost is lower and the efficiency is higher than that of the CBA protocol. Since the CBA/TBA suite of protocols do not need a trusted party, entities in these protocols store more information. Nevertheless, Tzeng and Tzeng [30], have proven that the message complexity exchanged between entities the CBA/TBA protocols is less than that of previous UMTS schemes. Last but not least, an authentication architecture for ad hoc networks has been proposed by Jacob and Corsen [14]. Their scheme presents the formats of all messages together with protocols that are supposed to achieve authentication.



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Remove the culture medium Rinse the cell layer with 5 ml BSS (for 10-cm tissue culture dishes) Add 4 ml trypsin-EDTA and return the vessel to the incubator for 5 10 min Keep the time of exposure as brief as possible (See (e) and (f) below) When the majority of cells have become well rounded or have detached from the culture dish, stop the reaction by adding a volume of BCS equal to 1/2 the volume of the trypsin Draw up the cell suspension with a pipette and, with the same pipette, use the suspension to gently wash the remaining cells from the dish It is not necessary to remove all of the cells from the dish, as most of the non broblastoid cells (which are not likely to be MSCs) in these cultures are more trypsin resistant than the spindle-shaped cells.





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Thus trypsinization represents, along with separation of nucleated cells by density centrifugation and attachment of broblastic cells to plastic, an important component of the process of the selection of MSCs from the total marrow cell population Transfer the cell suspension from all of the cultures to an appropriate-size centrifuge tube or tubes In our laboratory, we prefer to keep groups of culture dishes from a single preparation segregated from one another throughout the subculture process as a form of insurance against contamination of the entire group, because we have found that low levels of contamination are not always easily detectable For example, a preparation consisting of 12 culture dishes might be divided into 4 groups of 3 dishes; we would attempt to ensure that there was no cross-contamination among the groups by using separate pipettes and tubes for each group Centrifuge the tubes at 400 g.

As indicated in the Introduction, hMSCs remain in an undifferentiated state through repeated subculture. They can, however, be induced to differentiate along osteogenic lines by exposure to dexamethasone and ascorbic acid, and to mineralize by the later addition of -glycerophosphate.

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Remove the supernatant with a pipette or other suitable device Resuspend the cell pellet in a suitable volume of DMEM-LG-10FB (usually 5 or 10 ml) Remove a sample of the suspension with a Pasteur pipette or micropipettor and count the cells with a hemocytometer Adjust the volume of medium as necessary, and seed the cells at 3500 to 4000 cells per cm2 Change the culture medium every 3 to 4 days Further subculture of hMSCs is conducted in essentially the same manner except for the following considerations: i) Subcultured hMSCs are evenly distributed on the tissue culture vessel surface and are not in colonies as for primary cultures Therefore, the key criterion for determining when the cells should be trypsinized is the degree of con uence; basically, hMSCs should be trypsinized before they become con uent.

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ii) Passaged hMSCs are more easily trypsinized than primary cultures, so exposure of these cells to trypsin is usually limited to 5 min..

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